CACNA2D2 Promotes Tumorigenesis by Stimulating Cell Proliferation and Angiogenesis

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 coding for a putative auxiliary subunit of voltage-dependent 13 Although the implications of calcium signaling in genetic calcium channels (α2δ2 subunit). This subunit was identified as 18 cardiovascular, neurological or metabolic diseases have been promoting apoptosis, and was therefore thought to be a tumor 13 known for years,1 it has only recently been admitted to also be suppressor gene. We have, in the present study, addressed the involved in processes leading to cancer development,2 such as question of whether the α2δ2 subunit could be involved in PCA unregulated cell growth, resistance to apoptosis, enhanced progression. We show that α2δ2 is expressed in prostate epithelial angiogenesis and invasion. Indeed, it has been shown that cells and tissues and that its expression is enhanced during cancer α δ calcium transport mechanisms (through pumps, exchangers or development. We demonstrate that 2 2 regulates calcium channels) or calcium targets (calcium-binding proteins, calcium- homeostasis, nuclear factor of activated T-cells (NFAT) activity dependent kinases) may be subject to remodeling or altered and cell proliferation. Furthermore, we carried out xenograft fi expression in cancer cells.3 Voltage-gated calcium channels have experiments on immuno-de cient nude mice and we showed that α δ been shown either to be overexpressed, such as in colon cancer 2 2-overexpressing LNCaP PCA cells are more tumorigenic than α δ (Cav1.2 channels4) or downregulated (for instance, in lung cancer control LNCaP cells. Finally, we propose that the action of 2 2on (Cav3.1 channel5)) during cancer progression. tumor development probably occurs through a stimulation of Calcium channels are implicated in cancer development in both proliferation and angiogenesis. 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 RESULTS previously shown that voltage-dependent Cav3.2 T-type calcium α2δ2 expression in prostate tissues and cell lines channels are overexpressed in prostate cancer (PCA) cell lines, We have investigated whether α2δ (CACNA2Dx) proteins could be progressing toward a more aggressive phenotype.9 These expressed in human PCA cell lines and tissues. As shown in channels are expressed in human PCA acini and allow the Figure 1A, PCA LNCaP cells express the α2δ2 transcript. The other secretion of paracrine factors that may participate in cancer α2δ subunits investigated here (α2δ1 and α2δ3) are not expressed progression.12 Moreover, auxiliary voltage-dependent calcium in LNCaP cells. Other prostate cell lines express the α2δ2 transcript channels subunits are potentially involved in tumor growth. (DU145, Figure 1 and PC3; supplementary Figure S1A). Similarly, Indeed, several putative calcium channel subunit genes have been α2δ2 transcripts are present in both normal and cancerous human identified as being either down- or upregulated in some cancer prostate tissues (Figures 1B and C). Immuno-fluorescence (IF) tissues such as CACNA2D2 and CACNA2D3 in lung and gastric experiments showed that LNCaP cells express the α2δ2 subunit cancers,13–15 CACNB3 in recurrent non-small cell lung cancer16 or (Figure 1D). Immuno-staining for α2δ2 is particularly evident on involved in resistance of gliomas to chemotherapy (CACNG4).17 the cell periphery, close to plasma membrane area. Similar results A cluster of genes in locus 3p21 has been identified as a tumor were obtained with DU145 and PC3 cells (supplementary Figure S1B). suppressor gene cluster. In this cluster lies a CACNA2D2 gene 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 5384 at the expected size of 160 kD for α2δ2 (supplementary Figure S2). Immuno-histochemical (IH) staining and IF were carried out on The expression of this band was significantly reduced when LNCaP paraffin-embedded and frozen (IF) sections of human prostate cells were treated with siRNA-targeting α2δ2 (si-α2δ2a and tissues, respectively. As illustrated in Figures 1E and F, there was si-α2δ2b, supplementary Figure S2), showing that the antibody a consistent staining of α2δ2 in epithelial glandular acini in both used here successfully detects the α2δ2 protein. non-cancerous and cancerous human prostate tissues. In addition, Oncogene (2015) 5383 – 5394 © 2015 Macmillan Publishers Limited CACNA2D2 stimulates tumor cell growth M Warnier et al 5385 Figures 1E and F show that there was a stronger staining on the and α2δ2 subunits,19 on LNCaP cell proliferation. Figure 3C shows apical membranes of the epithelium than on basolateral that gabapentin (100 μM) reduced cell proliferation by about 35% membranes. after 48 h incubation. Gabapentin-induced inhibition then slightly In order to evaluate potential disorders in the expression of decreased, falling to 20% after 4 days, which may be due to α2δ2 between normal and tumoral tissues, we carried out a gabapentin desensitization. Another α2δ2 ligand (pregabalin; 20 comparison of the α2δ2 transcript expression using an RT–PCR 100 μM ) also slightly but significantly reduced LNCaP cell approach in 18 PCA tissues and 20 non-cancerous prostate tissues. proliferation (10.4 ± 1.07% inhibition at 48 h, supplementary As illustrated on Figures 1B and C, α2δ2 is more frequently Figure S3C). In addition, two different α2δ2-overexpressing LNCaP expressed (significantly different, Po0.001, Χ2) in cancer tissues clones expressing similar levels of α2δ2 subunits (clones C9 and (95% of cancer tissues expressed α2δ2) than in non-cancerous C11, supplementary Figure S2) displayed faster rates of prolifera- tissues (only 40% of non-cancerous prostate tissues expressed tion than did control LNCaP cells (Figure 3D), with a doubling time α2δ2). of 47.5 h for LNCaP against 40.5 h for clone 9 and 36 h for clone 11. Similarly, we compared the expression of α2δ2 protein on We then carried out IF experiments using Ki-67 staining (Figure 3E) prostate tissue microarray (TMA) in 24 different tissues (16 cancers on LNCaP and LNCaP-α2δ2 cells (clone C11). Ki-67 is a marker of and 8 non-cancerous tissues). As shown in Figure 2, epithelial cells proliferating cells. It is only expressed in phases G1, S, G2 and M, lining the acini were stained by the α2δ2 antibody in normal, whereas cells in the G0 phase do not express significant levels of benign prostatic hyperplasia and cancer samples. In advanced Ki-67.21 It can either exhibit punctuated nuclear localization in the cancers, glandular structure was, as expected, progressively lost G1 and S phases or a more homogeneous localization in the and stained cells were disseminated throughout the cancer tissue nucleus during the G2 and M phases. We demonstrate here that (see Figure 2B, Gleason Scores 4+5 and metastasis). Both stained α2δ2 is associated with a decrease in the number of cells in the surface and 3,3'-diaminobenzidine density increased in advanced G0 phase of the cell cycle. In our experiments, non-proliferating grades of PCA as compared with normal or hyperplasic prostate. cells represent 35.6 ± 1.6% of the whole-LNCaP cell population, Cellular localization differs between normal, benign prostatic whereas they represent only 21.6 ± 3.5% of the whole-LNCaP-α2δ2 hyperplasia, early stages of cancers and advanced cancers. In cell population. This correlates with an increase in the number normal, benign prostatic hyperplasia tissues and early stages of of cells in the G1/S and G2/M phases of the cell cycle in LNCaP- cancer (well-differentiated cancers: Gleason Scores o6), staining α2δ2 cells. Altogether, these results show that α2δ2 enhances is mostly apical and intracellular, whereas in advanced stages of prostate cell proliferation. cancer (from intermediately differentiated cancers—Gleason – — Scores 6 7(3+4) to poorly differentiated cancers, Gleason Scores Role of α2δ2 in calcium homeostasis from 7(4+3) and higher), staining increases in every cellular As α2δ2 has been shown to modulate voltage-dependent calcium compartment and remarkably on cell nuclei (Figure 2C).

Load more