CACNA2D2 promotes tumorigenesis by stimulating cell ... - Nature

Oncogene (2015) 34, 5383–5394 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

ORIGINAL ARTICLE

CACNA2D2 promotes tumorigenesis by stimulating cell proliferation and angiogenesis M Warnier, M Roudbaraki, S Derouiche, P Delcourt, A Bokhobza, N Prevarskaya and P Mariot In the present study, we have assessed whether a putative calcium channel α2δ2 auxiliary subunit (CACNA2D2 gene) could be involved in prostate cancer (PCA) progression. We therefore carried out experiments to determine whether this protein is expressed in PCA LNCaP cells and in PCA tissues, and whether its expression may be altered during cancer development. In addition, we evaluated the influence on cell proliferation of overexpressing or downregulating this subunit. In vitro experiments show that α2δ2 subunit overexpression is associated with increased cell proliferation, alterations of calcium homeostasis and the recruitment of a nuclear factor of activated T-cells pathway. Furthermore, we carried out in vivo experiments on immuno-deficient nude mice in order to evaluate the tumorigenic potency of the α2δ2 subunit. We show that α2δ2-overexpressing PCA LNCaP cells are more tumorigenic than control LNCaP cells when injected into nude mice. In addition, gabapentin, a ligand of α2δ2, reduces tumor development in LNCaP xenografts. Finally, we show that the action of α2δ2 on tumor development occurs not only through a stimulation of proliferation, but also through a stimulation of angiogenesis, via an increased secretion of vascular endothelial growth factor in cells overexpressing α2δ2. Oncogene (2015) 34, 5383–5394; doi:10.1038/onc.2014.467; published online 26 January 2015

INTRODUCTION Although the implications of calcium signaling in genetic cardiovascular, neurological or metabolic diseases have been known for years,1 it has only recently been admitted to also be involved in processes leading to cancer development,2 such as unregulated cell growth, resistance to apoptosis, enhanced angiogenesis and invasion. Indeed, it has been shown that calcium transport mechanisms (through pumps, exchangers or channels) or calcium targets (calcium-binding proteins, calciumdependent kinases) may be subject to remodeling or altered expression in cancer cells.3 Voltage-gated calcium channels have been shown either to be overexpressed, such as in colon cancer (Cav1.2 channels4) or downregulated (for instance, in lung cancer (Cav3.1 channel5)) during cancer progression. Calcium channels are implicated in cancer development in different tissues or organs, including prostate, breast, brain.6,7 Indeed, they have been demonstrated to participate in cell invasion, migration, differentiation or proliferation.8–11 We have previously shown that voltage-dependent Cav3.2 T-type calcium channels are overexpressed in prostate cancer (PCA) cell lines, progressing toward a more aggressive phenotype.9 These channels are expressed in human PCA acini and allow the secretion of paracrine factors that may participate in cancer progression.12 Moreover, auxiliary voltage-dependent calcium channels subunits are potentially involved in tumor growth. Indeed, several putative calcium channel subunit genes have been identified as being either down- or upregulated in some cancer tissues such as CACNA2D2 and CACNA2D3 in lung and gastric cancers,13–15 CACNB3 in recurrent non-small cell lung cancer16 or involved in resistance of gliomas to chemotherapy (CACNG4).17 A cluster of genes in locus 3p21 has been identified as a tumor suppressor gene cluster. In this cluster lies a CACNA2D2 gene

coding for a putative auxiliary subunit of voltage-dependent calcium channels (α2δ2 subunit).13 This subunit was identified as promoting apoptosis,18 and was therefore thought to be a tumor suppressor gene.13 We have, in the present study, addressed the question of whether the α2δ2 subunit could be involved in PCA progression. We show that α2δ2 is expressed in prostate epithelial cells and tissues and that its expression is enhanced during cancer development. We demonstrate that α2δ2 regulates calcium homeostasis, nuclear factor of activated T-cells (NFAT) activity and cell proliferation. Furthermore, we carried out xenograft experiments on immuno-deficient nude mice and we showed that α2δ2-overexpressing LNCaP PCA cells are more tumorigenic than control LNCaP cells. Finally, we propose that the action of α2δ2 on tumor development probably occurs through a stimulation of both proliferation and angiogenesis. RESULTS α2δ2 expression in prostate tissues and cell lines We have investigated whether α2δ (CACNA2Dx) proteins could be expressed in human PCA cell lines and tissues. As shown in Figure 1A, PCA LNCaP cells express the α2δ2 transcript. The other α2δ subunits investigated here (α2δ1 and α2δ3) are not expressed in LNCaP cells. Other prostate cell lines express the α2δ2 transcript (DU145, Figure 1 and PC3; supplementary Figure S1A). Similarly, α2δ2 transcripts are present in both normal and cancerous human prostate tissues (Figures 1B and C). Immuno-fluorescence (IF) experiments showed that LNCaP cells express the α2δ2 subunit (Figure 1D). Immuno-staining for α2δ2 is particularly evident on the cell periphery, close to plasma membrane area. Similar results were obtained with DU145 and PC3 cells (supplementary Figure S1B). In western blot experiments, we observed the presence of a band

Laboratoire de Physiologie Cellulaire, INSERM U1003, Villeneuve d'Ascq Cédex, France. Correspondence: Dr P Mariot, Laboratoire de Physiologie Cellulaire, INSERM U1003, Bâtiment SN3, Université Lille1, Villeneuve d'Ascq Cédex 59655, France. E-mail: [email protected] Received 9 July 2014; revised 5 November 2014; accepted 19 December 2014; published online 26 January 2015

CACNA2D2 stimulates tumor cell growth M Warnier et al

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at the expected size of 160 kD for α2δ2 (supplementary Figure S2). The expression of this band was significantly reduced when LNCaP cells were treated with siRNA-targeting α2δ2 (si-α2δ2a and si-α2δ2b, supplementary Figure S2), showing that the antibody used here successfully detects the α2δ2 protein.

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Immuno-histochemical (IH) staining and IF were carried out on paraffin-embedded and frozen (IF) sections of human prostate tissues, respectively. As illustrated in Figures 1E and F, there was a consistent staining of α2δ2 in epithelial glandular acini in both non-cancerous and cancerous human prostate tissues. In addition,

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Figures 1E and F show that there was a stronger staining on the apical membranes of the epithelium than on basolateral membranes. In order to evaluate potential disorders in the expression of α2δ2 between normal and tumoral tissues, we carried out a comparison of the α2δ2 transcript expression using an RT–PCR approach in 18 PCA tissues and 20 non-cancerous prostate tissues. As illustrated on Figures 1B and C, α2δ2 is more frequently expressed (significantly different, P o 0.001, Χ2) in cancer tissues (95% of cancer tissues expressed α2δ2) than in non-cancerous tissues (only 40% of non-cancerous prostate tissues expressed α2δ2). Similarly, we compared the expression of α2δ2 protein on prostate tissue microarray (TMA) in 24 different tissues (16 cancers and 8 non-cancerous tissues). As shown in Figure 2, epithelial cells lining the acini were stained by the α2δ2 antibody in normal, benign prostatic hyperplasia and cancer samples. In advanced cancers, glandular structure was, as expected, progressively lost and stained cells were disseminated throughout the cancer tissue (see Figure 2B, Gleason Scores 4+5 and metastasis). Both stained surface and 3,3'-diaminobenzidine density increased in advanced grades of PCA as compared with normal or hyperplasic prostate. Cellular localization differs between normal, benign prostatic hyperplasia, early stages of cancers and advanced cancers. In normal, benign prostatic hyperplasia tissues and early stages of cancer (well-differentiated cancers: Gleason Scores o6), staining is mostly apical and intracellular, whereas in advanced stages of cancer (from intermediately differentiated cancers—Gleason Scores 6–7(3+4)—to poorly differentiated cancers, Gleason Scores from 7(4+3) and higher), staining increases in every cellular compartment and remarkably on cell nuclei (Figure 2C). We also analyzed staining density using color deconvolution to extract 3,3'-diaminobenzidine staining from counterstaining (Azure blue). We show that 3,3'-diaminobenzidine density, and thus α2δ2 expression, was significantly enhanced in PCA tissues as compared with non-cancerous tissues (Figure 2C). Furthermore, cytosolic staining was significantly increased between grades 2 and 3, showing a correlation between α2δ2 expression and cancer progression (Figure 2Cb). α2δ2 regulates cell proliferation Different approaches were used to evaluate the role of α2δ2 in prostate cell proliferation (Figure 3). First, LNCaP cell proliferation was reduced by siRNA-targeting α2δ2 (50 nM si-α2δ2a or si-α2δ2b) as measured either by MTS assay (Figure 3A) or manual counting (supplementary Figure S3A). Kinetic experiments (Figure 3A) indeed illustrate that both si-α2δ2 had a significant inhibitory effect on cell proliferation only after 3–4 days’ incubation and reached their maximal action after 5–7 days (inducing a 40–80% reduction in cell proliferation). Treatment of DU145 and PC3 cells with si-α2δ2 similarly reduced proliferation (supplementary Figure S3B). Fluorescence-activated cell sorting analysis of cell cycle shows that si-α2δ2 increased the number of LNCaP cells in G1 phase and decreased the proportion of cells in G2/M and S phases (Figure 3B). We therefore tested gabapentin, a ligand of α2δ1

and α2δ2 subunits,19 on LNCaP cell proliferation. Figure 3C shows that gabapentin (100 μM) reduced cell proliferation by about 35% after 48 h incubation. Gabapentin-induced inhibition then slightly decreased, falling to 20% after 4 days, which may be due to gabapentin desensitization. Another α2δ2 ligand (pregabalin; 100 μM20) also slightly but significantly reduced LNCaP cell proliferation (10.4 ± 1.07% inhibition at 48 h, supplementary Figure S3C). In addition, two different α2δ2-overexpressing LNCaP clones expressing similar levels of α2δ2 subunits (clones C9 and C11, supplementary Figure S2) displayed faster rates of proliferation than did control LNCaP cells (Figure 3D), with a doubling time of 47.5 h for LNCaP against 40.5 h for clone 9 and 36 h for clone 11. We then carried out IF experiments using Ki-67 staining (Figure 3E) on LNCaP and LNCaP-α2δ2 cells (clone C11). Ki-67 is a marker of proliferating cells. It is only expressed in phases G1, S, G2 and M, whereas cells in the G0 phase do not express significant levels of Ki-67.21 It can either exhibit punctuated nuclear localization in the G1 and S phases or a more homogeneous localization in the nucleus during the G2 and M phases. We demonstrate here that α2δ2 is associated with a decrease in the number of cells in the G0 phase of the cell cycle. In our experiments, non-proliferating cells represent 35.6 ± 1.6% of the whole-LNCaP cell population, whereas they represent only 21.6 ± 3.5% of the whole-LNCaP-α2δ2 cell population. This correlates with an increase in the number of cells in the G1/S and G2/M phases of the cell cycle in LNCaPα2δ2 cells. Altogether, these results show that α2δ2 enhances prostate cell proliferation. Role of α2δ2 in calcium homeostasis As α2δ2 has been shown to modulate voltage-dependent calcium channel activity and plasma membrane expression,22 we studied whether α2δ2 could regulate calcium homeostasis in LNCaP cells. Though not decreased by si-α2δ2 (Figure 4a), the cytosolic-free calcium concentration was increased by α2δ2 overexpression, in either 2 or 10 mM extracellular calcium concentrations (Figure 4b). Furthermore, to assay whether calcium homeostasis, and internal calcium store capacity, was altered in LNCaP-α2δ2 cells, we challenged the cells with ionomycine (1 μM), a Ca2+ ionophore able to induce calcium mobilization from endoplasmic reticulum stores23 and thapsigargin (0.5 μM), an endoplasmic reticulum Ca2+ ATPase inhibitor.24 Our results show that both ionomycine and thapsigargin led to slightly stronger calcium releases in LNCaPα2δ2 cells than in LNCaP-ctl cells (Figures 4c and d). On the contrary, si-α2δ2 lessened the calcium release induced by either thapsigargin or ionomycine (Figure 4d). This demonstrates that α2δ2 regulates calcium homeostasis in LNCaP cells. We thus investigated whether the NFAT-calcineurin pathway could be involved in α2δ2 signaling. First, we show that NFAT activity was greater in LNCaP-α2δ2 than in LNCaP-ctl cells (Figure 4e). To evaluate the role of the calcineurin/NFAT pathway in cell proliferation, we used two different calcineurin inhibitors (cyclosporin A and FK506), which both reduced LNCaP cell proliferation. Reduction of cell proliferation by these inhibitors of calcineurin was more pronounced in LNCaP-α2δ2 cells than in LNCaP-ctl cells (Figure 4f).

Figure 1. Expression of α2δ2 in prostate cells and tissues. (A) Prostate cancer LNCaP and DU145 cell lines were screened by RT–PCR for the different members of the α2δ family. Only the α2δ2 transcript is detected in LNCaP and DU145 cells at the expected size of 505 bp. Human brain is used as a positive control. (B) Human prostate tissues were screened for the expression of α2δ2. Among the prostate samples tested (n = 20 for non-cancer tissues, n = 18 for cancer tissues), α2δ2 is more frequently expressed in cancer tissues (***P o0.001, Χ2-test). (C) Examples of RT–PCR performed on non-cancer and cancer tissues (numbered from 1 to 10: N1 to N10 for normal and C1 to C10 for cancer). (D) Confocal immuno-fluorescence experiments showing the expression of α2δ2 in LNCaP cells (×20 objective). Staining is particularly pronounced at the cell periphery. (E) Confocal immuno-fluorescence experiment showing the expression of α2δ2 in prostate hyperplasic tissues (a and b: different magnifications of the same field). (F) Confocal immuno-fluorescence experiments showing the expression of α2δ2 in prostate cancer tissues. Panel a shows a cancer (grade 3) with small acini still observable. Panel b is an enlargment of the acinus labeled in panel a. In panel c is shown a cancer (grade 4) where acini are fused and lumen is not observable. Bar size: 40 μm. Lum: lumen of the acini. Arrow: apical side of the epithelium. Diamond headed arrow: basal side. © 2015 Macmillan Publishers Limited

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Figure 2. Immuno-histochemical study of α2δ2 expression in normal and diseased prostate. (A and B) Overview of prostate TMA showing different normal, hyperplasic and cancer tissues. (A) × 5 objective, (B) × 40 objective). Gleason Scores are indicated on each picture. M: rectal metastasis, BPH: hyperplasia, N: normal prostate tissue. Immuno-histochemical staining against α2δ2 was revealed using peroxidase-DAB reaction. (C) Color images were deconvoluted, and intensity of cell staining was analyzed and converted into optical density (OD). (a) As illustrated, OD is enhanced in cancer tissues in acini surrounding cells. (b) Histogram showing the average OD for different Gleason grades in cytosol and in nucleus. The number of cells analyzed for each bar was comprised between 143 and 882. OD was compared between normal and other conditions (*P o0.05, **P o0.01, ***P o0.001) and between grade 2 and other conditions (#Po 0.05, ##P o0.01, ###Po 0.001). Comparison was assessed with Student–Newman–Keuls tests.

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Figure 3. α2δ2 promotes cell proliferation in prostate cancer LNCaP cell lines. (A-a) A treatment with siRNA (50 nM)-targeting α2δ2 (si-α2δ2a) inhibits LNCaP cell growth as measured with MTS assay. Kinetic experiments show that inhibition by si-α2δ2a reaches its peak (40%) 6 days after the onset of treatment. (b) LNCaP cell proliferation was significantly reduced by two different siRNA-targeting α2δ2(si-α2δ2a and si-α2δ2b, 50 nM) 6 days after treatment. (B) FACS analysis shows that treatments with si-α2δ2a increased the proportion of cells in G0/G1 phase, whereas decreased the proportion of cells in the S and G2/M phases. (C) Gabapentin (100 μM), a ligand of α2δ2 reduces cell proliferation as measured by MTS assay. (D) Kinetics of cell proliferation in two different clones of LNCaP cells overexpressing α2δ2 (LNCaP-α2δ2-C9 and LNCaP-α2δ2-C11). (E-a) Bar representation of the proportion of LNCaP cells in G0, G1/S or G2/M phase as measured by Ki-67 staining in LNCaPctl and LNCaP-α2δ2-C11 cells. (b) Example of Ki-67 immuno-staining. The overexpression of α2δ2 reduces the number of cells in the G0 phase, whereas increases the number of cells in the G1/S phases. (*Po0.05, **P o0.01, ***P o0.001).

Overexpression of α2δ2 promotes in vivo tumor development, blood vessel formation and VEGF secretion Immunodeficient nude mice were injected on both flanks with either LNCaP-ctl cells, LNCaP-Neo cells or with LNCaP-α2δ2 cells (Figure 5). Though we did not compare the level of expression of α2δ2 between human prostate tumors and LNCaP-α2δ2 clones, this last model was particularly useful to determine the role of α2δ2 in proliferation and tumor growth. Indeed, the increased expression of α2δ2 in LNCaP-α2δ2 clones may qualitatively reflect the increased expression of α2δ2 occurring in advanced PCA. Tumor size, which was measured twice a week, usually began to © 2015 Macmillan Publishers Limited

increase after 4–7 weeks (Figure 5b). The latent period was a little bit longer in LNCaP-α2δ2 tumors (49.5 ± 3, 34.5 ± 3.5, 37.8 ± 2.8 days for LNCaP-α2δ2, LNCaP-ctl and LNCaP-Neo, respectively, Figure 5c). Once the tumors were detectable, they grew with a doubling time of 12.2 ± 1.9 days in LNCaP tumors and 10.7 ± 1.2 days for LNCaP-Neo tumors to reach an average size of 940 ± 146 mm3 (1.26 ± 0.22 g). For their part, LNCaP-α2δ2 tumors grew with a doubling time of 8.3 ± 0.8 days to reach a size of 1760 ± 154 mm3 (2.3 ± 0.21 g) 11 weeks after cell injection (Figure 5d). In different tumors, maximum tumor size was usually reached 4 weeks after the onset of tumor growth. Oncogene (2015) 5383 – 5394


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Figure 4. Calcium imaging studies show that α2δ2 is involved in calcium homeostasis. (a) Downregulating α2δ2 expression in LNCaP cells (using siRNA-targeting α2δ2 (si-α2δ2a and si-α2δ2b, 50 nM)) did not change the basal cytosolic calcium concentration in both 2 and 10 mM external calcium concentration conditions. (b) Overexpressing α2δ2 in LNCaP cells (LNCaP-α2δ2) slightly increased the basal cytosolic calcium concentration in both 2 and 10 mM external calcium concentration conditions. (c) Application of 0.5 μM Thaspigargin (TG) induced a calcium release, as shown by the increase in the F340/F380 ratio, of lower amplitude in LNCaP-ctl cells as compared with LNCaP-α2δ2 cells. (d) Average peak amplitude of calcium release induced by thapsigargin (TG, 0.5 μM) and ionomycine (iono 1 μM) in LNCaP-ctl, LNCaP-α2δ2 cells and in LNCaP cells treated with si-α2δ2a, si-α2δ2b or si-ctl. (e) Measurement of NFAT activity in LNCaP cells shows that the overexpression of α2δ2 is associated with a significant stimulation of NFAT activity. (f) Inhibitors of calcineurin (cyclosporin A (CsA 1 and 5 μM) and FK506 (10 μM)) reduced cell growth stimulated by the overexpression of α2δ2. (*P o0.05, **P o0.01, ***P o0.001).

Altogether, LNCaP-α2δ2 tumors grew faster and reached a larger size than LNCaP-ctl and LNCaP-Neo tumors. We also assessed the effect of gabapentin on nude mice bearing LNCaP-ctl tumors. As seen in Figure 5e, gabapentin consumption (via drinking water) from the onset of the experiment considerably slowed tumoral development. Both LNCaP-ctl and LNCaP-α2δ2 tumors (Figures 5a and 6) were abundantly irrigated and characterized by signs of hemorrhages with leakage of red blood cells in surrounding tissues and numerous foci of inflammation and necrosis (Figures 6A and B). We did not detect any significant differences in necrosis between LNCaP-ctl and LNCaP-α2δ2 tumors. On the contrary, proliferating cell nuclear antigen IF studies, which allow the identification of cells in the S phase of the cell cycle, show that there are more proliferating cells in LNCaP-α2δ2 tumors than in LNCaP-ctl tumors (Figure 6C). Oncogene (2015) 5383 – 5394

In addition, histological slides show that there are more blood vessels in LNCaP-α2δ2 tumors than in LNCaP-ctl tumors (Figures 6Aa and Ab). We thus stained blood vessels with CD31 (Figures 7A and B), which is a common marker for endothelial cells and used to detect angiogenesis in tumors. We show that there is a twofold increase in the surface stained by CD31 in LNCaP-α2δ2 tumors as compared with LNCaP-ctl tumors (Figure 7C). This was confirmed using western blots experiments, which demonstrate that there is a 50% increase in CD31 expression in LNCaP-α2δ2 tumors (Figure 7D). As α2δ2 is associated with blood vessel formation, we measured vascular endothelial growth factor (VEGF) expression and secretion in prostate cells and tumors. As shown in Figure 7D, western blot experiments demonstrate that VEGF expression was slightly but significantly increased in tumors overexpressing α2δ2. In addition, as shown in Figure 7E, enzyme-linked immunosorbant © 2015 Macmillan Publishers Limited


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Figure 5. α2δ2 potentiates in vivo tumor growth. (a) Mice were injected in both flanks with LNCaP, LNCaP-Neo or LNCaP-α2δ2 cells. After killing of the animals, tumors were extracted from the animals, weighed and prepared for RNA or protein extraction or immuno-histochemistry. (b) Kinetics of tumor growth in both control and α2δ2 tumors. (c) Bar plot of the latency period before the development of detectable tumors (4100 mm3) in LNCaP-ctl, LNCaP-Neo and LNCaP-α2δ2 tumors. (d) Average doubling time was lower in LNCaP-α2δ2 tumors than in LNCaPcontrol and LNCaP-Neo tumors. (e) Administration of Gabapentin via drinking water (400 mg/l) from the onset and throughout the experiment reduced the average kinetic of tumor development in animals injected with LNCaP-ctl cells. (*P o0.05, **P o0.01, ***P o0.001).

assay tests show that overexpression of α2δ2 significantly enhanced in vitro VEGF secretion in two different clones of LNCaP-α2δ2 cells. Implication of α2δ2 presence in other tissues In order to assess the potential relevance of α2δ2 in tumorigenesis in other tissues, we compared the expression of α2δ2 on TMA in other cancer tissues (lung, breast and colon). We show that α2δ2 is consistently expressed in lung, breast and colon cancer tissues (two cases for each cancer) and that stained surface is greater in cancer vs normal tissues (Figure 8a). In addition, we carried out RT–PCR in various pancreatic cell lines. Our results show that α2δ2 is expressed in pancreatic tumorigenic carcinoma cell lines (ASPC1, BxPC3, Capan1, MiaPaca2, Panc1), but not in the human non-tumorigenic immortalized pancreatic ductal H6c7 epithelial cell line (Figure 8b). DISCUSSION We demonstrate for the first time in this study that α2δ2, a putative calcium channel accessory subunit,25 is able to promote cell proliferation in vitro and tumorigenesis in vivo. We first show that α2δ2 is expressed in PCA cell lines and tissues and that α2δ2 is more frequently expressed in cancer tissues than in noncancerous prostate tissues obtained from surgical human samples. We demonstrate that α2δ2 is expressed mainly in the prostate epithelium and that its expression is higher in cancer tissues than in normal tissues. This result was unexpected because other publications had previously shown that the CACNA2D2 gene, coding for the α2δ2 protein, is located in a tumor suppressor gene cluster13 (the 3p21.3 chromosomal region). This region may be deleted or hypermethylated in various cancers, such as lung or pancreatic cancers.14 In addition, CACNA2D2 promotes apoptosis in non-small cell lung cancer cells18 and reduces tumor growth in nude mice. However, some studies have shown that CACNA2D2 © 2015 Macmillan Publishers Limited

itself is not hypermethylated. In a recent study, it was even shown that CACNA2D2 is overexpressed in breast cancer cell lines compared with non-tumorigenic cells.26 As we show that it may be overexpressed in a variety of cancer cells (prostate, breast, pancreas, lung, colon), CACNA2D2 may possess oncogenic properties. Interestingly, another protein of the same family, the α2δ1 subunit (CACNA2D1) has recently been shown to be expressed in liver cancer stem cells and that RNAi knockdown of α2δ1 expression reduces tumorigenicity in cancerous liver cells,27 suggesting that different proteins of the CACNA2D family may have oncogenic properties. The CACNA2D2 gene may therefore, according to the tissues or circumstances, behave as a tumor suppressor gene or an oncogene. Such a property is known for various genes, such as REST (repressor element-1 silencing transcription factor), which acts as an oncogene in neural cells or a tumor suppressor gene in breast or lung cells,28 or other transcription factors such as WT1, KLF4, SnoN and Runx (for review see reference 28). The RASFF1 gene has also been reported to display both oncogenic and tumor suppressive properties in lung neuroendocrine tumors,29 depending on the isoform expressed. Interestingly, the RASFF1 gene is located in the same 3p21.3 locus as CACNA2D2. In vitro experiments were carried out using various proliferation assays, and all the methods used here demonstrate that α2δ2 activates proliferation of PCA cells, decreasing the number of cells in the Go and G1 phases and increasing the proportion of cells in the S and M phases. This was shown using overexpression studies, siRNA strategy and ligand-inhibition of α2δ2. Furthermore, we demonstrate that α2δ2 overexpression is associated with tumor growth, VEGF secretion in vitro and blood vessel formation in vivo. We thus suggest that α2δ2, through a stimulation of VEGF secretion, promotes angiogenesis. Therefore, both proliferative and angiogenic actions of α2δ2 may be responsible for α2δ2induced tumor development in mice xenografts. In addition, we show that gabapentin, a known α2δ2 ligand, reduced tumor size Oncogene (2015) 5383 – 5394


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Figure 6. Histology of LNCaP tumors. (A) Low magnification views (×5 objective) of a control LNCaP tumor (a) and an LNCaP-α2δ2 tumor (b). (B) Different views of a control LNCaP tumor (a) and an LNCaP-α2δ2 tumor (c) at higher magnification (×20). Numerous foci of necrosis and inflammation or hemorrhages could be observed in tumors ((b) LNCaP and (d) LNCaP-α2δ2). (C) PCNA immuno-fluorescence (a) and quantification of the percentage of PCNA-positive nuclei normalized to the total number of nuclei (DAPI) (b). Red arrows: examples of hemorrhages and leakage of red blood cells. Blue arrows: inflammatory cells. Black arrows: necrosis sites. (*P o0.05).

in mice xenografts, confirming that α2δ2 may be involved in tumor development. It has been proposed that α2δ2 is a voltage-dependent calcium channel accessory subunit and could regulate both targeting and activity of α1 pore subunits.30,31 As PCA cells express functional Cav3.2 T-type calcium channels,9 α2δ2 involvement in tumorigenesis could rely on its interaction with Cav3.2 channels. Furthermore, we have shown previously that Cav3.2 overexpression Oncogene (2015) 5383 – 5394

enhances cell growth, whereas its downregulation leads to reduced proliferation.32 We show here that the α2δ2 expression regulates calcium homeostasis. As Cav3.2 and α2δ2 exert similar actions on both calcium signaling and cell growth, they may be involved in a common pathway, leading to increases in cell proliferation, through a possible interaction. However, various interacting domains, including a large C-terminal sequence that protrudes into the extracellular environment, have been © 2015 Macmillan Publishers Limited


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Figure 7. α2δ2 promotes blood vessel formation in xenografted LNCaP tumors through VEGF secretion. (A) Immuno-fluorescent identification of CD31 (a): CD31 (green), (b): DAPI (blue), (c): overlay of DAPI and CD31. (B) Immuno-histochemical (peroxydase/DAB) staining of CD31 (top panel: LNCaP tumor, bottom panel: LNCaP-α2δ2 tumor. (C) CD31-positive area in LNCaP and LNCaP-α2δ2 tumors. (D-d) Examples of western blots of CD31 and VEGF in eight different tumors (LNCaP tumors: T-LNCaP, LNCaP-α2δ2 tumors: T-α2δ2) from four different mice (Mx). (b) Relative density of CD31 bands (normalized to β-actin) observed in western blots (mean ± s.e.m. from 10 tumors). (c) Relative density of VEGF bands (normalized to β-actin) observed in western blots (mean ± s.e.m. from 10 tumors). (e) VEGF secretion measured using ELISA assay in LNCaP cells and LNCaP cells overexpressing α2δ2 (clones C9 and C11). Overexpression of α2δ2 was associated with an increased secretion of VEGF.


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MATERIALS AND METHODS Cell culture and human tissues LNCaP, DU145 and PC3 PCA cell lines were purchased from the American Type Culture Collection and cultured in a humidified atmosphere at 37 °C (95% air—5% CO2) as described by Gackière et al.12 in RPMI 1640 (GIBCO, Life Technologies, St Aubin, France), supplemented with 10% fetal bovine serum (Sigma, St Quentin Fallavier, France), and 2 mM L-glutamine (Sigma). LNCaP cells stably expressing CACNA2D2 (LNCaP-α2δ2) or transfected with an empty pcDNA3 plasmid (LNCaP-Neo) were generated as described in supplementary methods. Pancreatic cell lines (H6C7, ASPC1, BxPC3, Capan1, MiaPaca2, Panc1) used to detect the expression of CACNA2D2 were obtained and cultured as described by Kondratska et al.38 Paraffin-embedded TMAs (from prostate (Figure 2), lung, colon and breast (Figure 8)) were obtained from US Biomax (Rockville, MD, USA). Human brain cDNA were purchased from Amsbio (Abington, UK). Prostate tissue samples (prostate adenocarcinoma and hyperplasia) were obtained from consenting patients, following the local ethical considerations and as described in supplementary methods. Unless specified, all products were purchased from Sigma.

Cell transfection Two small RNAs (Eurogentec, Angers, France) interfering with the human coding sequence of CACNA2D2 (Genebank accession # NM_001174051) were designed and referred to as si-α2δ2a (5′-GACCAACGUUCUGAUCUGC (dTdT)-3′) and si-α2δ2b (5'-AACAAGGUCAACUAUUCAUAC(dTdT)-3'). The siRNAs used in this study included a nonspecific control siRNA against Luciferase (5′-CUUACGCUGAGUACUUCGA (dTdT)-3′accession # FN554878). Cells were transfected with 50 nM siRNA using HiPerFect Transfection Reagent (Qiagen, Courtaboeuf, France) as described previously.12 The efficiency of the siRNA was validated by western blot analysis (supplementary Figure S2).

Analysis of the α2δ2 subunit gene expression by RT–PCR Figure 8. Expression of α2δ2 in non-prostatic cancer tissues (breast, lung and colon). (a) Immuno-histochemical staining against α2δ2 was revealed using peroxidase-DAB reaction in breast, lung and colon cancer and normal tissues (×5 objective). Grade and stage are indicated on the right corner of each tissue microarray. (b) RT–PCR carried out in six pancreatic cell lines, a human immortalized, nontumorigenic pancreatic ductal epithelial cell line, H6c7 and five tumorigenic carcinoma cell lines ASPC1, BxPC3, Capan1, MiaPaca2, Panc1. α2δ2 transcript was present only in carcinoma cell lines.

demonstrated in α2δ2, which would suggest that α2δ2 may interact with proteins other than calcium channels. For example, the Von Willebrand A domain,33 in the extracellular portion of α2δ2, is also present in proteins such as integrins involved in the interaction with the extracellular matrix.34 The adhesion of LNCaP cells on the extracellular matrix could thus be affected by the overexpression of α2δ2 and this could delay tumor onset as observed in our experiments. This extracellular domain may also serve as a receptor to extracellular ligands such as thrombospondin 1.35,36 Thrombospondin have themselves been shown to control angiogenesis through their interaction with their numerous receptors.37Although thrombospondin 1 may inhibit angiogenesis through their interaction with CD36 or VLDL-R membrane receptors, they can also promote angiogenesis after binding with α3β1 integrins.37 It is therefore conceivable that α2δ2 promotes cell proliferation or angiogenesis through a signaling pathway, which does not involve voltage-dependent calcium channels. We will thus go on to investigate whether α2δ2 subunits could regulate angiogenesis in response to thrombospondin 1 binding. Altogether, our results suggest that α2δ2 is a potentially relevant marker of PCA progression and could therefore be evaluated as relevant target in diagnostic assays. In addition, our experiments show that molecular or pharmacological inhibition of α2δ2 leads to a reduction of tumor growth, suggesting its relevance as a basis for the development of alternative PCA treatments. Oncogene (2015) 5383 – 5394

RNA extraction was carried out using Trizol according to the method originally described by Chomczynski and Sacchi.39 RT–PCR was carried out as previously described.12 The PCR primers in this study were: 5′-TGCAAGAAGACCTTGTCACA-3′ (forward) and 5′-ACACAATTGTTGAG CCCTCA-3′ (reverse) for the 400 bp CACNA2D1 amplicon (accession #: GI 179761), 5′-GATGCGGAGCTAGAGGATGA-3′ (forward) and 5′-GCTGTACGTG TTGAGATGCT-3′ (reverse) for the 505 bp CACNA2D2 amplicon (accession #: BC 152438), 5′-CCATGGAGGTGAAGAAGACA -3′ (forward) and 5′-TTGT CACCAGCTTTCAGGAA-3′ (reverse) for the 500/439 bp CACNA2D3 amplicon (accession #: BC NM_018398), and 5′-CAGAGCAAGAGAGGCATCCT-3′ (forward) and 5′-GTTGAAGGTCTCAAACATGAT-3′ (reverse) for the 220 bp β-actin amplicon (accession #:: NM_001101).

Western blotting After washing in phosphate-buffered saline, cells were collected in a lysis buffer (Triton X-100 1%, Na deoxycholate, 1%, NaCl 150 mM, PO4NaK 10 mM, pH 7.2), with an anti-protease cocktail and incubated on ice for 45 min. The lysates were centrifuged at 12 000 g for 10 min at 4 °C. The protein concentration of the supernatant was determined by the BCA assay (Pierce Chemical Biology, Courtaboeuf, France). Western-blot analysis was performed as described elsewhere12 using the following primary antibodies: anti-CACNA2D2 (130 kDa, Santa Cruz sc-66822, Heidelberg, Germany; 1/200 in WB, 1/100 in IF/IH), antiCACNA2D2 (Abnova H00009254-M12, Walnut, CA, USA; 1/50 in IF/IH), antiproliferating cell nuclear antigen (36 kDa, Santa Cruz sc-56, 1/200 in WB, 1/100 in IF), anti-Ki-67 (Santa Cruz sc101861, 1/100 in IF/IH), anti-CD31 (130 kDa, Abcam, Paris, France; ab28364, 1/200 in WB, 1/50 in IF/IH), anti-VEGF (43 kDa, Abcam ab46154, 1/200 in WB, 1/50 in IF/IH), anti-β-actin (43 kDa, Sigma A5441, 1/4000 in WB), anti-calnexin (95 kDa, Millipore MAB3126, Guyancourt, France; 1/2000 in WB). Each experiment was repeated at least three times.

Immuno-staining and confocal analysis The protein expression studies of PCA cells and tissues were carried out using indirect IF analysis on acetone-fixed cells or frozen section of prostate and immuno-histochemistry (IH) on formalin-fixed paraffinembedded tissues (TMA). TMA (US Biomax) and mice tumoral tissues were studied using IH procedures as previously described.40 Frozen sections of human prostate were prepared for IF experiments as described previously.12 © 2015 Macmillan Publishers Limited


5393 Calcium imaging Calcium imaging was carried out in Hank's Balanced Salt Solution containing 142 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2 or 10 mM CaCl2, 10 mM HEPES and 5.6 mM glucose. The osmolarity and pH of external solutions were adjusted to 310 mOsm l − 1 and 7.4, respectively. Cytosolic Ca2+ concentration was measured and analyzed using Fura2-loaded cells (2 μM) as described elsewhere.41

multiple comparisons), and Χ2-tests to compare proportions. Differences were considered significant with *Po0.05; **Po0.01; ***Po 0.001.

Declaration of approval for animal studies In vivo experiments were conducted on mice according to the agreement provided by the local ethical comity (protocol CEEA 202012).

NFAT assay

CONFLICT OF INTEREST

Cells were seeded in 60-mm dishes and were transfected with a NFATluciferase plasmid or with luciferase only (1 μg of plasmid per dish). Cells were lysed and proteins extracted using the Luciferase Assay System Kit (Promega, Madison, WI, USA). NFAT-luciferase activity was then measured according to the manufacturer’s protocol. NFAT activity was normalized to the number of cells measured using a MTS assay. Each experiment was repeated at least three times.

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank the members of the imaging platform BICEL, E Richard and C Slommiany for their helpful contribution. This work was supported by INSERM, the University of Lille1 and the Region Nord-Pas de Calais. This work was supported by INSERM, the University of Lille1 and the Region Nord-Pas de Calais.

Cell proliferation and cell cycle analysis Cell viability was assessed by a colorimetric MTS assay (CellTiter 96) according to manufacturer’s instructions. Cell proliferation was also assessed by manual counting of cells extruding Trypan Blue. Ki-67 or proliferating cell nuclear antigen staining were also used to discriminate cells in quiescence (G0 phase) and cycling cells. For cell cycle analysis, cells were grown in three 60-mm dishes. After treatment, cells were harvested and treated as described previously32 with propidium iodide (25 μg/ml). Propidium iodide fluorescence was measured at 520 nm (excitation at 488 nm) using a flow cytometer (Beckman Coulter Epics XL4-MCL, Roissy CDG, France). Data were interpreted using Multicycle for Windows (Phoenix Flow system). Each experiment was repeated at least three times.

VEGF secretion assay Secretion of VEGF was measured in vitro by enzyme-linked immunosorbant assay as described by the manufacturer (Abcam). Cells were cultured in 12-well plates for 3 days before the culture medium was changed. VEGF was assayed in the culture medium after 24 h’s incubation (three wells per condition). Each experiment was repeated at least three times.

In vivo experiments Six-week-old male swiss nude mice (Charles River Laboratories, St Germain sur l'Arbresle, France) were injected with LNCaP, LNCaP-Neo or LNCaPα2δ2 cells. Six million cells were injected into both flanks of each mouse. Such double-injection protocols are often used in xenograft assays.42,43 Though one cannot exclude that a tumor may affect the other, LNCaP cells injected subcutaneously do not metastasize 12 weeks after injection44 and should not diffuse to the other tumor. We did not observe any sign of metastasis. Cells were prepared in a mixture composed of 50% phosphatebuffered saline and 50% BD-Matrigel (BD Bioscience, Rungis, France). Tumors were measured twice a week using a caliper, and animals were killed 12 weeks after injection unless the mice had to be killed earlier if the total tumor size reached 10% of the animal weight. Tumor volume was calculated using the following formula: Volume (in mm3) = length (in mm) × depth (in mm) × width (in mm) × π/6. On the day of killing, tumors were weighed and divided for further IH, western blot and RT–PCR experiments. At least 10 animals per condition were used. In order to study gabapentin action on tumor development, gabapentin was administered via the drinking water, which was renewed every other day. Water consumption was assessed to be 5 ml/day/mouse. Gabapentin concentration in the drinking water was set at 400 mg/l in order for each animal to consume 60 mg/day/kg, which is to the maximal dose used in human medical treatments. The stability of gabapentin being very high in water even at room temperature, its concentration should not be affected in our experiments.45

Statistical analysis Plots were produced using Origin 7.0 (Microcal Software Inc., Northampton, MA, USA). Results are expressed as mean ± s.e.m. Statistical analyses were performed using unpaired t-tests (for comparing two groups), analysis of variance tests followed by either Dunnett (for multiple control vs test comparisons) or Student–Newman–Keuls post-tests (for © 2015 Macmillan Publishers Limited

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2015) 5383 – 5394
