TRPC6 channels are required for proliferation, migration and invasion of breast cancer cell lines by modulation of Orai1 and Orai3 surface exposure

cancers Article TRPC6 Channels Are Required for Proliferation, Migration and Invasion of Breast Cancer Cell Lines by Modulation of Orai1 and Orai3 Surface Exposure Isaac Jardin 1,†,* , Raquel Diez-Bello 1,†, Jose J. Lopez 1, Pedro C. Redondo 1, Ginés M. Salido 1, Tarik Smani 2and Juan A. Rosado 1,* 1Cellular Physiology Research Group, Department of Physiology, Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain; [email protected] (R.D.-B.); [email protected] (J.J.L.); [email protected] (P.C.R.); [email protected] (G.M.S.) 2Department of Medical Physiology and Biophysic, Institute of Biomedicine of Sevilla, 41013 Sevilla, Spain; [email protected] *Correspondence: [email protected] (I.J.); [email protected] (J.A.R.); Tel.: +34-927257100 (ext. 51376) (I.J. & J.A.R.); Fax: +34-927257110 (I.J. & J.A.R.) † These authors contributed equally to this work. Received: 5 August 2018; Accepted: 13 September 2018; Published: 14 September 2018   Abstract: Transient receptor potential channels convey signaling information from a number of stimuli to a wide variety of cellular functions, mainly by inducing changes in cytosolic Ca 2+ concentration. Different members of the TRPC, TRPM and TRPV subfamilies have been reported to play a role in tumorigenesis. Here we show that the estrogen receptor positive and triple negative breast cancer cell lines, MCF7 and MDA-MB-231, respectively, exhibit enhanced expression of the TRPC6 channel as compared to the non-tumoral MCF10A cell line. In vitro TRPC6 knockdown using shRNA impaired MCF7 and MDA-MB-231 cell proliferation, migration and invasion detected by BrdU incorporation, wound healing and Boyden chamber assays, respectively. Using RNAi-mediated TRPC6 silencing as well as overexpression of the pore-dead dominant-negative TRPC6 mutant we have found that TRPC6 plays a relevant role in the activation of store-operated Ca 2+ entry in the breast cancer cell lines but not in non-tumoral breast cells. Finally, we have found that TRPC6 interacts with Orai1 and Orai3 in MCF7 and MDA-MB-231 cells and is required for the translocation of Orai1 and Orai3 to the plasma membrane in MDA-MB-231 and MCF7 cells, respectively, upon Ca 2+ store depletion. These findings introduce a novel mechanism for the modulation of Ca 2+ influx and the development of different cancer hallmarks in breast cancer cells. Keywords: TRPC6; Orai1; Orai3; store-operated calcium entry; MCF7; MDA-MB-231 1. Introduction Breast cancer is among the leading causes of cancer death in women worldwide, accounting for about 25% of all diagnosed female cancers [ 1 ]. Breast cancer cells are characterized by a high proliferation rate, resistance to programmed cell death, and increased capability to migrate and invade surrounding tissues [ 2 ]. These hallmarks can develop through different mechanisms that lead to the onset and progression of breast cancer, among them the alteration in the PI3K pathway [ 3 ], abnormal activation of the MAPK signaling [4] or anomalous intracellular Ca2+ signaling [5]. Cytosolic free-Ca 2+ concentration is a crucial factor for a variety of cellular processes [ 6 ] and a number of genes encoding ion channels have been found among those altered in cancer cells [ 7 ]. There is a growing body of evidence supporting the relevant role of ion channels, and particularly Ca 2+ Cancers 2018,10, 331; doi:10.3390/cancers10090331 www.mdpi.com/journal/cancers Cancers 2018,10, 331 2 of 18 channels, in the mechanisms underlying cell growth and proliferation, migration, apoptosis resistance and angiogenesis in cancer cells. Among the Ca 2+ channels in cancer cells, Orai1, the pore-forming subunit of the Ca 2+ release-activated Ca 2+ (CRAC) channel [ 8 , 9 ], which is the best characterized store-operated Ca 2+ channel, has been found to be overexpressed in the human cancer cells investigated, including breast cancer [ 10 ], melanoma [ 11 ], clear cell renal carcinoma [ 12 ] and non-small cell lung carcinoma [ 13 ], except in prostate cancer cells, whose expression has been reported to be reduced as compared to normal tissue [ 14 ]. The information concerning the Orai1 homologs Orai2 and Orai3 is rather scarce but Orai2 has been found to be overexpressed in parathyroid adenoma [ 15 ] and acute myeloid leukemia cells [ 16 ], while Orai3 is overexpressed in estrogen receptor-expressing (ER + ) breast cancer cell lines [ 17 ] and in prostate cancer tissue specimens obtained from resection surgeries as compared to noncancerous tissue [18]. On the other hand, transient receptor potential (TRP) channels, especially certain members of the TRPC, TRPM and TRPV subfamilies, have also been reported to play a relevant role in the progression of different types of cancer. Among them, TRPV6 is overexpressed in a number of cancer cell types and participates in the progression of prostate cancer [ 19 ], acquiring its oncogenic potential via Orai1/TRPC1-dependent translocation to the plasma membrane [ 20 ]. TRPM8 regulates the motility of a variety of cancer cells including oral squamous carcinoma, lung cancer or prostate cancer cells [ 21 ], where its plasma membrane localization and tumorigenic potential are regulated by TRP channel-associated factors [ 22 ]. Studies concerning TRPC subfamily members have mainly focused on TRPC1, whose involvement in tumorigenesis varies depending on the stage and type of cancer considered [ 21 , 23 ]. TRPC6 has been reported to play a relevant role in the proliferation of gastric [ 24 ], prostate [ 25 ], esophageal squamous cell carcinoma [ 26 ] and hepatome cells [ 27 ]. Furthermore, TRPC6 is required for migration and invasion of hepatocellular carcinoma cells [ 28 ]. TRPC6 channels have been shown to be overexpressed in human breast ductal adenocarcinoma compared to non-tumoral tissue [ 29 , 30 ] and both, TRPC3 and TRPC6, have been reported to be significantly up-regulated in breast cancer biopsies compared to normal tissue [ 31 ]; however, the molecular basis of the functional role of TRPC6 in breast cancer cells and its involvement in the cancer hallmarks remains unclear. Here we show that TRPC6 is required for proliferation, migration and invasion of the ER + cell line MCF7 and the triple negative MDA-MB-231 cell line. Silencing TRPC6 protein expression, as well as overexpression of a pore-dead dominant-negative TRPC6 mutant has revealed that TRPC6 plays an important role in the activation of store-operated Ca 2+ entry (SOCE) in both MCF7 and MDA-MB-231 cell lines, which is likely mediated by the role of TRPC6 in the translocation to the plasma membrane of Orai3 or Orai1, respectively, in the cell lines investigated. 2. Results 2.1. TRPC6 Is Overexpressed in MCF7 and MDA-MB-231 Breast Cancer Cell Lines and is Required for Breast Cancer Cell Proliferation, Migration and Invasion Consistent with the previous study by Aydar and coworkers [ 31 ], Western blot analysis of whole cell lysates from the non-tumoral breast MCF10A cell line, the ER + and triple negative breast cancer cell lines MCF7 and MDA-MB-231, respectively, with a specific anti-human TRPC6 antibody revealed that the expression of this protein is relatively low in the non-tumoral cell line (Figure 1). Furthermore, TRPC6 expression in the MCF7 and MDA-MB-231 cell lines is significantly greater (approximately 350 and 460%, respectively) than in non-tumoral cells. TRPC6 expression in the different cell lines, normalized to the β -actin content and expressed as percentage of the expression level in MCF10A, is shown in Figure 1(bar graphs; n = 6). We have further explored the involvement of TRPC6 in the ability of MCF10A, MCF7 and MDA-MB-231 to proliferate. To address this issue, cells transfected with shTRPC6 or shRNA control vector (shRNAcv), were subjected to the BrdU cell proliferation assay. Cancers 2018,10, 331 3 of 18 Cancers 2018, 10, 331 3 of 18 Figure 1. Cellular expression of TRPC6 in non-tumoral and breast cancer cell lines. MCF10A, MCF7 and MDA-MB-231 cells were lysed and subjected to Western blotting with anti-TRPC6 antibody, followed by reprobing with anti-β-actin antibody for protein loading control. Bar graphs represent TRPC6 expression normalized to the β-actin content and expressed as percentage of the TRPC6 expression in non-tumoral MCF10A cells. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. * p < 0.05 compared to TRPC6 expression in MCF10A cells. As shown in Figure 2a, cell transfection with shTRPC6 significantly attenuated TRPC6 expression in MCF10A, MCF7 and MDA-MB-231 cells (p < 0.05; n = 6). Next, we explored the effect of transfection with shTRPC6 in cell proliferation in the three cell lines. Forty-eight hours after transfection (time = 0h), as well as 24, 48 and 72h later, cell proliferation was assessed. As expected, the shTRPC6 was without effect in MCF10A proliferation, which is consistent with the low native TRPC6 expression and indicates a lack of effect of shTRPC6 in cell proliferation in this cell line (Figure 2b; n = 6). Interestingly, silencing TRPC6 protein expression significantly attenuated MCF7 and MDAMB-231 cell proliferation at all the times investigated as compared to cells transfected with shRNAcv (Figure 2b; p < 0.05; n = 4). Therefore, our observations reveal that TRPC6 is essential for ER+ and triple negative breast cancer cell proliferation. Next, we assessed the relevance of TRPC6 in the ability of these cell lines to migrate. MCF10A, MCF7 and MDA-MB-231 cells were subjected to the well-established wound healing assay. Cells were seeded, scratched, and cultured in medium supplemented with 1% serum to prevent further cell growth. Migration of cells was quantitated as described in Materials and Methods. To explore the role of TRPC6 in cell migration MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or control plasmid and cell migration was evaluated. As shown in Figure 3a, MCF10A, MCF7 and MDA-MB-231 cells transfected with shRNAcv significantly reduced the wound size during the first 48 h (p < 0.05; n = 3). TRPC6 expression silencing did not affect the ability of MCF10A to migrate (Figure 3a; n = 3), which is consistent with the low expression of TRPC6 in this cell line. Interestingly, silencing TRPC6 expression significantly attenuated MCF7 and MDA-MB-231 migration as compared to cells transfected with shRNAcv (Figure 3a; p < 0.05; n = 3), which indicates that TRPC6 plays an important role in MCF7 and MDA-MB-231 cell migration. We have further investigated the role of TRPC6 in in vitro invasion analysed using the transwell migration assay. After transfection with shRNAcv, a significant amount of MCF7 and MDA-MB-231 cells, especially the latter, passed across the transwell insert (Figure 3b). We even found a large number of MDA-MB-231 cells adhered to the surface of the lower chamber (Figure 3b, bottom panel). By contrast, we were unable to detect MCF10A cells in the undersurface of the transwell insert [32]. Interestingly, as depicted in Figure 3b, a lesser number of MCF7 and MDA-MB-231 cells were able to migrate to the undersurface of the transwell insert upon TRPC6 expression silencing as compared to cells treated with control shRNA (p < 0.05; n = 5). Consistently, the number of invasive MDA-MB-231 cells attached to the surface of the lower chamber was clearly reduced after transfection with shTRPC6 (Figure 3b, bottom panel). Figure 1. Cellular expression of TRPC6 in non-tumoral and breast cancer cell lines. MCF10A, MCF7 and MDA-MB-231 cells were lysed and subjected to Western blotting with anti-TRPC6 antibody, followed by reprobing with antiβ -actin antibody for protein loading control. Bar graphs represent TRPC6 expression normalized to the β -actin content and expressed as percentage of the TRPC6 expression in non-tumoral MCF10A cells. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. * p< 0.05 compared to TRPC6 expression in MCF10A cells. As shown in Figure 2a, cell transfection with shTRPC6 significantly attenuated TRPC6 expression in MCF10A, MCF7 and MDA-MB-231 cells (p< 0.05; n = 6). Next, we explored the effect of transfection with shTRPC6 in cell proliferation in the three cell lines. Forty-eight hours after transfection ( time = 0 h ), as well as 24, 48 and 72 h later, cell proliferation was assessed. As expected, the shTRPC6 was without effect in MCF10A proliferation, which is consistent with the low native TRPC6 expression and indicates a lack of effect of shTRPC6 in cell proliferation in this cell line (Figure 2b; n = 6). Interestingly, silencing TRPC6 protein expression significantly attenuated MCF7 and MDA-MB-231 cell proliferation at all the times investigated as compared to cells transfected with shRNAcv (Figure 2b; p< 0.05; n = 4). Therefore, our observations reveal that TRPC6 is essential for ER + and triple negative breast cancer cell proliferation. Next, we assessed the relevance of TRPC6 in the ability of these cell lines to migrate. MCF10A, MCF7 and MDA-MB-231 cells were subjected to the well-established wound healing assay. Cells were seeded, scratched, and cultured in medium supplemented with 1% serum to prevent further cell growth. Migration of cells was quantitated as described in Materials and Methods. To explore the role of TRPC6 in cell migration MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or control plasmid and cell migration was evaluated. As shown in Figure 3a, MCF10A, MCF7 and MDA-MB-231 cells transfected with shRNAcv significantly reduced the wound size during the first 48 h (p< 0.05; n = 3). TRPC6 expression silencing did not affect the ability of MCF10A to migrate (Figure 3a; n = 3), which is consistent with the low expression of TRPC6 in this cell line. Interestingly, silencing TRPC6 expression significantly attenuated MCF7 and MDA-MB-231 migration as compared to cells transfected with shRNAcv (Figure 3a; p< 0.05; n = 3), which indicates that TRPC6 plays an important role in MCF7 and MDA-MB-231 cell migration. We have further investigated the role of TRPC6 in in vitro invasion analysed using the transwell migration assay. After transfection with shRNAcv, a significant amount of MCF7 and MDA-MB-231 cells, especially the latter, passed across the transwell insert (Figure 3b). We even found a large number of MDA-MB-231 cells adhered to the surface of the lower chamber (Figure 3b, bottom panel). By contrast , we were unable to detect MCF10A cells in the undersurface of the transwell insert [ 32 ]. Interestingly, as depicted in Figure 3b, a lesser number of MCF7 and MDA-MB-231 cells were able to migrate to the undersurface of the transwell insert upon TRPC6 expression silencing as compared to cells treated with control shRNA (p< 0.05; n = 5). Consistently, the number of invasive MDA-MB-231 cells attached to the surface of the lower chamber was clearly reduced after transfection with shTRPC6 (Figure 3b, bottom panel). Cancers 2018,10, 331 4 of 18 Cancers 2018, 10, 331 4 of 18 Figure 2. TRPC6 expression is required for MCF7 and MDA-MB-231 cell proliferation. (a) MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or shRNA control vector (shRNAcv), as indicated. After 48h cells were lysed and subjected to Western blotting with anti-TRPC6 antibody, followed by reprobing with anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. (b) MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or scramble plasmid and 48 h later cell proliferation was assessed for a further 24, 48 and 72 h using the BrdU cell proliferation assay kit, as described in the Material and Methods. Bar graphs represent cell proliferation 0, 24, 48 and 72 h after cell transfection, presented as BrdU uptake rate. * p < 0.05 compared to the corresponding control (cells transfected with shRNAcv). Figure 3. Cont. Figure 2. TRPC6 expression is required for MCF7 and MDA-MB-231 cell proliferation. ( a ) MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or shRNA control vector (shRNAcv), as indicated. After 48h cells were lysed and subjected to Western blotting with anti-TRPC6 antibody, followed by reprobing with antiβ -actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. (b) MCF10A , MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or scramble plasmid and 48 h later cell proliferation was assessed for a further 24, 48 and 72 h using the BrdU cell proliferation assay kit, as described in the Material and Methods. Bar graphs represent cell proliferation 0, 24, 48 and 72 h after cell transfection, presented as BrdU uptake rate. * p< 0.05 compared to the corresponding control (cells transfected with shRNAcv). Cancers 2018, 10, 331 4 of 18 Figure 2. TRPC6 expression is required for MCF7 and MDA-MB-231 cell proliferation. (a) MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or shRNA control vector (shRNAcv), as indicated. After 48h cells were lysed and subjected to Western blotting with anti-TRPC6 antibody, followed by reprobing with anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. (b) MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or scramble plasmid and 48 h later cell proliferation was assessed for a further 24, 48 and 72 h using the BrdU cell proliferation assay kit, as described in the Material and Methods. Bar graphs represent cell proliferation 0, 24, 48 and 72 h after cell transfection, presented as BrdU uptake rate. * p < 0.05 compared to the corresponding control (cells transfected with shRNAcv). Figure 3. Cont. Figure 3. Cont. Cancers 2018,10, 331 5 of 18 Cancers 2018, 10, 331 5 of 18 Figure 3. Role of TRPC6 in breast cancer cell migration and invasion. MCF10A, MCF7 and MDA-MB231 cells were transfected with shTRPC6 or control shRNAcv. Forty-eight hours after transfection cells were subjected to wound healing assay (a) or transwell migration assay (b) as described in Methods. (a) Images were acquired at 0 and 48 h from the beginning of the assay. The dotted lines define the areas lacking cells. The bar graphs represent the wound size, in micrometers, at the different conditions, expressed as the mean ± SEM of three independent experiments. * p < 0.05 compared to the time = 0 h. § p < 0.05 compared to the corresponding time in shRNAcv transfected cells. (b) Images show the stained cells as obtained from the transwell migration assay subjected to the different experimental conditions. The bar graphs represent the percentage of cell invasion as compared to MDA-MB-231 cells transfected with shRNAcv, expressed as the mean ± SEM of five independent experiments. * p < 0.05 compared to the corresponding shRNAcv transfected cells. Bottom panels show representative pictures of the invasive cells adhered to the bottom of the lower chamber. Figure 3. Role of TRPC6 in breast cancer cell migration and invasion. MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or control shRNAcv. Forty-eight hours after transfection cells were subjected to wound healing assay ( a ) or transwell migration assay ( b ) as described in Methods. ( a ) Images were acquired at 0 and 48 h from the beginning of the assay. The dotted lines define the areas lacking cells. The bar graphs represent the wound size, in micrometers, at the different conditions, expressed as the mean ± SEM of three independent experiments. * p< 0.05 compared to the time = 0 h. § p< 0.05 compared to the corresponding time in shRNAcv transfected cells. ( b ) Images show the stained cells as obtained from the transwell migration assay subjected to the different experimental conditions. The bar graphs represent the percentage of cell invasion as compared to MDA-MB-231 cells transfected with shRNAcv, expressed as the mean ± SEM of five independent experiments. * p< 0.05 compared to the corresponding shRNAcv transfected cells. Bottom panels show representative pictures of the invasive cells adhered to the bottom of the lower chamber. Cancers 2018,10, 331 6 of 18 We confirmed the role of TRPC6 in breast cancer cell migration and proliferation by expressing a pore-dead dominant-negative TRPC6 (TRPC6dn) mutant. As shown in Figure 4a, expression of the TRPC6dn mutant significantly reduced MCF7 and MDA-MB-231 migration as compared to cells transfected with empty vector (p< 0.05; n = 3). Cancers 2018, 10, 331 6 of 18 We confirmed the role of TRPC6 in breast cancer cell migration and proliferation by expressing a pore-dead dominant-negative TRPC6 (TRPC6dn) mutant. As shown in Figure 4a, expression of the TRPC6dn mutant significantly reduced MCF7 and MDA-MB-231 migration as compared to cells transfected with empty vector (p < 0.05; n = 3). Figure 4. Expression of TRPC6dn mutant attenuates cell migration and proliferation in breast cancer cells. (a) MCF7 and MDA-MB-231 cells were transfected with TRPC6dn expression plasmid or empty vector (mock), as indicated. Forty-eight hours after transfection cells were subjected to wound healing assay as described in Methods. Images were acquired at 0 and 48 h from the beginning of the assay. The dotted lines define the areas lacking cells. The bar graphs represent the wound size, in micrometers, at the different conditions, expressed as the mean ± SEM of three independent experiments. * p < 0.05 compared to the time = 0h. § p < 0.05 compared to the corresponding time in mock-treated cells. (b) MCF7 and MDA-MB-231 cells were transfected with TRPC6dn expression plasmid or empty vector (mock), as indicated, and 48 h later cell proliferation was assessed for a further 24, 48 and 72 h using the BrdU cell proliferation assay kit, as described in the Material and Methods. Bar graphs represent cell proliferation 0, 24, 48 and 72 h after cell transfection, presented as BrdU uptake rate. * p < 0.05 compared to the corresponding control (mock-transfected cells). Figure 4. Expression of TRPC6dn mutant attenuates cell migration and proliferation in breast cancer cells. ( a ) MCF7 and MDA-MB-231 cells were transfected with TRPC6dn expression plasmid or empty vector (mock), as indicated. Forty-eight hours after transfection cells were subjected to wound healing assay as described in Methods. Images were acquired at 0 and 48 h from the beginning of the assay. The dotted lines define the areas lacking cells. The bar graphs represent the wound size, in micrometers, at the different conditions, expressed as the mean ± SEM of three independent experiments. *p< 0.05 compared to the time = 0 h. § p< 0.05 compared to the corresponding time in mock-treated cells. (b) MCF7 and MDA-MB-231 cells were transfected with TRPC6dn expression plasmid or empty vector (mock), as indicated, and 48 h later cell proliferation was assessed for a further 24, 48 and 72 h using the BrdU cell proliferation assay kit, as described in the Material and Methods. Bar graphs represent cell proliferation 0, 24, 48 and 72 h after cell transfection, presented as BrdU uptake rate. * p< 0.05 compared to the corresponding control (mock-transfected cells). Cancers 2018,10, 331 7 of 18 Furthermore, expression of the TRPC6dn mutant significantly attenuated MCF7 and MDA-MB-231 cell proliferation at all the times investigated as compared to cells transfected with empty vector (Figure 4b; p< 0.05; n = 3). These findings confirm that TRPC6 is required for MCF7 and MDA-MB-231 breast cancer cells migration and proliferation. 2.2. Functional Role of TRPC6 in SOCE in Breast Cancer Cell Lines As our results indicate that TRPC6 knockdown significantly attenuates relevant features of cancer cells, such as proliferation, migration and in vitro invasion, we have explored the possible mechanism underlying the functional role of TRPC6 in these cells. SOCE has been reported to play an important role supporting several cancer hallmarks [ 16 , 33 , 34 ]. Hence, we have evaluated whether TRPC6 plays a role in the activation of SOCE in breast cancer cells by transfecting non-tumoral MCF10A and cancer MCF7 and MDA-MB-231 cells with shTRPC6 or shRNAcv, as control. As depicted in Figure 5a–c, in cells transfected with shRNAcv suspended in a Ca 2+ -free medium, treatment with the SERCA inhibitor TG (1 µ M) resulted in a transient increase in cytosolic free-Ca 2+ concentration due to Ca 2+ release from the intracellular Ca 2+ stores. Subsequent addition of CaCl 2 (1 mM) to the extracellular medium resulted in a further increase in cytosolic free-Ca 2+ concentration indicative of SOCE. TG-induced Ca 2+ release was similar in all the cell lines investigated while Ca 2+ influx was significantly greater in MDA-MB-231 cells (Figure 5g,h; p< 0.05; n = 40 cells/day/3–5 days). Attenuation of TRPC6 expression by cell transfection with shTRPC6 significantly inhibited SOCE in MCF7 and MDA-MB-231 cells by 70%, without having any effect on Ca 2+ release from the intracellular stores (Figure 5a–c,g–h; p< 0.05). Transfection of MCF10A cells with shTRPC6 did not significantly alter TG-induced Ca 2+ release or entry, which is consistent with the low TRPC6 expression at the protein level in these cells. Altogether these findings indicate that TRPC6 plays a relevant role in the activation of SOCE in MCF7 and MDA-MB-231 breast cancer cells while this protein has not a detectable role in non-tumoral MCF10A cells. Cancers 2018, 10, 331 7 of 18 Furthermore, expression of the TRPC6dn mutant significantly attenuated MCF7 and MDA-MB231 cell proliferation at all the times investigated as compared to cells transfected with empty vector (Figure 4b; p < 0.05; n = 3). These findings confirm that TRPC6 is required for MCF7 and MDA-MB231 breast cancer cells migration and proliferation. 2.2. Functional Role of TRPC6 in SOCE in Breast Cancer Cell Lines As our results indicate that TRPC6 knockdown significantly attenuates relevant features of cancer cells, such as proliferation, migration and in vitro invasion, we have explored the possible mechanism underlying the functional role of TRPC6 in these cells. SOCE has been reported to play an important role supporting several cancer hallmarks [16,33,34]. Hence, we have evaluated whether TRPC6 plays a role in the activation of SOCE in breast cancer cells by transfecting non-tumoral MCF10A and cancer MCF7 and MDA-MB-231 cells with shTRPC6 or shRNAcv, as control. As depicted in Figure 5a–c, in cells transfected with shRNAcv suspended in a Ca2+-free medium, treatment with the SERCA inhibitor TG (1 µM) resulted in a transient increase in cytosolic free-Ca2+ concentration due to Ca2+ release from the intracellular Ca2+ stores. Subsequent addition of CaCl2 (1 mM) to the extracellular medium resulted in a further increase in cytosolic free-Ca2+ concentration indicative of SOCE. TG-induced Ca2+ release was similar in all the cell lines investigated while Ca2+ influx was significantly greater in MDA-MB-231 cells (Figure 5g,h; p < 0.05; n = 40 cells/day/3-5 days). Attenuation of TRPC6 expression by cell transfection with shTRPC6 significantly inhibited SOCE in MCF7 and MDA-MB-231 cells by 70%, without having any effect on Ca2+ release from the intracellular stores (Figures 5a–c and 4g–h; p < 0.05). Transfection of MCF10A cells with shTRPC6 did not significantly alter TG-induced Ca2+ release or entry, which is consistent with the low TRPC6 expression at the protein level in these cells. Altogether these findings indicate that TRPC6 plays a relevant role in the activation of SOCE in MCF7 and MDA-MB-231 breast cancer cells while this protein has not a detectable role in non-tumoral MCF10A cells. Figure 5. Cont. Figure 5. Cont. Cancers 2018,10, 331 8 of 18 Cancers 2018, 10, 331 8 of 18 Figure 5. TRPC6 is required for store-operated Ca2+ entry in breast cancer cell lines. (a–c) MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or scramble plasmid (shRNAcv), as indicated. Forty-eight hours after transfection, fura-2-loaded cells were perfused with a Ca2+-free medium (100 µM EGTA added) and then stimulated with TG (1 µM) followed by reintroduction of external Ca2+ (final concentration 1 mM) to initiate Ca2+ entry. Data are mean ± SEM of 40 cells/day/35 days. (d–f) MCF7 and MDA-MB-231 cells were transfected with TRPC6dn mutant expression plasmid or empty vector (mock), as indicated. After 48 h cells were lysed and subjected to western blotting with anti-TRPPC6 antibody, followed by reprobing with anti-β-actin antibody for protein loading control (d). Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. (e and f) Forty-eight hours after transfection, fura-2-loaded cells were perfused with a Ca2+-free medium (100 µM EGTA added) and then stimulated with TG (1 µM) followed by reintroduction of external Ca2+ (final concentration 1 mM) to initiate Ca2+ entry. Data are mean ± SEM of 40 cells/day/3-5 days. Bar graphs represent TG-induced Ca2+ release (g) and entry (h) in MCF10A, MCF7 and MDA-MB-231 cells untreated or transfected with the indicated plasmids. Data are expressed as mean ± SEM and presented as percentage of control (MCF10A cells treated with scramble plasmid). * represents p < 0.05 as compared to scramble-treated MCF10A cells. § represents p < 0.05 as compared to the same cell line transfected with shRNAcv. In order to further explore whether the observed effect depends on cation entry through the channel or it is rather associated to a mechanism involving the expression of the protein itself, we overexpressed the TRPC6dn mutant in MCF7 and MDA-MB-231 cells and looked for its effect on TGinduced Ca2+ release and entry. As shown in Figure 5d, TRPC6dn was efficiently expressed in both cell types. As depicted in Figures 5e–h, overexpression of TRPC6dn in MCF7 and MDA-MB-231 cells significantly reduced TG-evoked Ca2+ entry to a similar extent to transfection of shTRPC6 (p < 0.05 as compared to control; n = 40 cells/day/3–5 days), which indicates that cation influx through TRPC6 plays an important role in SOCE in these cells. Overexpression of TRPC6dn also resulted in a significant decrease in the ability of MCF7 cells to accumulate Ca2+ into TG-sensitive stores (Figure 5e,g; p < 0.05; n = 40 cells/day/3–5 days), an effect that might be attributed to the inhibition of SOCE. Figure 5. TRPC6 is required for store-operated Ca 2+ entry in breast cancer cell lines. ( a – c ) MCF10A, MCF7 and MDA-MB-231 cells were transfected with shTRPC6 or scramble plasmid (shRNAcv), as indicated . Forty-eight hours after transfection, fura-2-loaded cells were perfused with a Ca 2+ -free medium (100 µ M EGTA added) and then stimulated with TG (1 µ M) followed by reintroduction of external Ca 2+ (final concentration 1 mM) to initiate Ca 2+ entry. Data are mean ± SEM of 40 cells/day/3–5 days . ( d – f ) MCF7 and MDA-MB-231 cells were transfected with TRPC6dn mutant expression plasmid or empty vector (mock), as indicated. After 48 h cells were lysed and subjected to western blotting with anti-TRPPC6 antibody, followed by reprobing with antiβ -actin antibody for protein loading control ( d ). Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. ( e and f ) Forty-eight hours after transfection, fura-2-loaded cells were perfused with a Ca 2+ -free medium (100 µ M EGTA added) and then stimulated with TG (1 µ M) followed by reintroduction of external Ca 2+ (final concentration 1 mM) to initiate Ca 2+ entry. Data are mean ± SEM of 40 cells/day/3–5 days. Bar graphs represent TG-induced Ca 2+ release ( g ) and entry ( h ) in MCF10A, MCF7 and MDA-MB-231 cells untreated or transfected with the indicated plasmids. Data are expressed as mean ± SEM and presented as percentage of control (MCF10A cells treated with scramble plasmid). * represents p< 0.05 as compared to scramble-treated MCF10A cells. § represents p< 0.05 as compared to the same cell line transfected with shRNAcv. In order to further explore whether the observed effect depends on cation entry through the channel or it is rather associated to a mechanism involving the expression of the protein itself, we overexpressed the TRPC6dn mutant in MCF7 and MDA-MB-231 cells and looked for its effect on TG-induced Ca 2+ release and entry. As shown in Figure 5d, TRPC6dn was efficiently expressed in both cell types. As depicted in Figure 5e–h, overexpression of TRPC6dn in MCF7 and MDA-MB-231 cells significantly reduced TG-evoked Ca 2+ entry to a similar extent to transfection of shTRPC6 ( p< 0.05 as compared to control; n = 40 cells/day/3–5 days), which indicates that cation influx through TRPC6 plays an important role in SOCE in these cells. Overexpression of TRPC6dn also resulted in a significant decrease in the ability of MCF7 cells to accumulate Ca 2+ into TG-sensitive stores (Figure 5e,g; p< 0.05; n = 40 cells/day/3–5 days), an effect that might be attributed to the inhibition of SOCE. Cancers 2018,10, 331 9 of 18 2.3. TRPC6 Expression Is Required for Plasma Membrane Localization of Orai1 and Orai3 in Breast Cancer Cells Breast cancer MCF7 and MDA-MB-231 cells have been reported to express both Orai1 and Orai3 channels. However, the relative expression level and function differs from ER + MCF7 cells to triple negative MDA-MB-231 cells [ 35 ]. While SOCE in MDA-MB-231 cells entirely depends on Orai1, MCF7 SOCE is mainly mediated by Orai3, whose expression, regulated by ER α [ 17 ], is predominant over that of Orai1 [ 35 ]. Our results confirm that Orai1 is overexpressed in the breast cancer cell lines and that Orai3 expression is significantly enhanced in MCF7 (Figure 6a; p< 0.05; n = 6), as previously reported [ 35 ]. In order to explore the mechanism underlying the sensitivity of SOCE to TRPC6 expression and function we have first investigated the interaction of TRPC6 with Orai1 and Orai3 by co-immunoprecipitation from MCF7 and MDA-MB-231 cell lysates. Resting and TG-treated cells were used for this study to determine whether Ca 2+ store depletion plays any role in the possible interaction between TRPC6 and the Orai proteins investigated. As shown in Figure 6b,c, immunoprecipitation of cell lysates with anti-TRPC6 antibody followed by Western blotting with anti-Orai1 or anti-Orai3 antibody reveals that TRPC6 interacts with both proteins in resting cells. Interestingly, our results suggest that in MCF7 cells the interaction of TRPC6 with Orai3 is apparently greater than with Orai1, and, conversely, in MDA-MB-231 cells, TRPC6 seems to interact predominantly with Orai1 over Orai3 (Figure 6b,c; n = 6). Although these apparent differences might be attributed to the use of two different antibodies, if we look at the association of TRPC6 with Orai1, whose expression we and others have found to be similar in MCF7 and MDA-MB-231 cells [ 35 ] (Figure 6a), and we normalize the data with the amount of TRPC6 pulled down, our results indicate that more Orai1 is bound to TRPC6 in MDA-MB-231 cells (p< 0.05; n = 6). In addition, we have found that the interaction of TRPC6 with Orai1 and Orai3 is not altered by treatment with 1 µ M TG for 3 min (Figure 6b,c), which, as depicted in Figure 5, is able to induce significant store depletion. Cancers 2018, 10, 331 9 of 18 2.3. TRPC6 Expression Is Required for Plasma Membrane Localization of Orai1 and Orai3 in Breast Cancer Cells Breast cancer MCF7 and MDA-MB-231 cells have been reported to express both Orai1 and Orai3 channels. However, the relative expression level and function differs from ER+ MCF7 cells to triple negative MDA-MB-231 cells [35]. While SOCE in MDA-MB-231 cells entirely depends on Orai1, MCF7 SOCE is mainly mediated by Orai3, whose expression, regulated by ER [17], is predominant over that of Orai1 [35]. Our results confirm that Orai1 is overexpressed in the breast cancer cell lines and that Orai3 expression is significantly enhanced in MCF7 (Figure 6a; p < 0.05; n = 6), as previously reported [35]. In order to explore the mechanism underlying the sensitivity of SOCE to TRPC6 expression and function we have first investigated the interaction of TRPC6 with Orai1 and Orai3 by co-immunoprecipitation from MCF7 and MDA-MB-231 cell lysates. Resting and TG-treated cells were used for this study to determine whether Ca2+ store depletion plays any role in the possible interaction between TRPC6 and the Orai proteins investigated. As shown in Figure 6b,c, immunoprecipitation of cell lysates with anti-TRPC6 antibody followed by Western blotting with anti-Orai1 or anti-Orai3 antibody reveals that TRPC6 interacts with both proteins in resting cells. Interestingly, our results suggest that in MCF7 cells the interaction of TRPC6 with Orai3 is apparently greater than with Orai1, and, conversely, in MDA-MB-231 cells, TRPC6 seems to interact predominantly with Orai1 over Orai3 (Figure 6b,c; n = 6). Although these apparent differences might be attributed to the use of two different antibodies, if we look at the association of TRPC6 with Orai1, whose expression we and others have found to be similar in MCF7 and MDA-MB-231 cells [35] (Figure 6a), and we normalize the data with the amount of TRPC6 pulled down, our results indicate that more Orai1 is bound to TRPC6 in MDA-MB-231 cells (p < 0.05; n = 6). 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