NFATc3 controls tumour growth by regulating proliferation and migration of human astroglioma cells

Calcium/Calcineurin/Nuclear Factor of Activated T cells (Ca/CN/NFAT) signalling pathway is the main calcium (Ca2+) dependent signalling pathway involved in the homeostasis of brain tissue.

Here, we study the presence of NFATc members in human glioma by using U251 cells and a collection of primary human glioblastoma (hGB) cell lines. We show that NFATc3 member is the

predominant member. Furthermore, by using constitutive active NFATc3 mutant and shRNA lentiviral vectors to achieve specific silencing of this NFATc member, we describe cytokines and

molecules regulated by this pathway which are required for the normal biology of cancer cells. Implanting U251 in an orthotopic intracranial assay, we show that specific NFATc3 silencing has

a role in tumour growth. In addition NFATc3 knock-down affects both the proliferation and migration capacities of glioma cells in vitro. Our data open the possibility of NFATc3 as a target

for the treatment of glioma.

Gliomas are among the most severe forms of cancer. Gliomas originate from glia cells (astrocytes, oligodendrocytes, ependymal cells) or cancer stem cells, and are classified by the World

Health Organization (WHO) into four grades based on malignancy (I to IV). The most common of these fatal tumours is grade IV astrocytoma or glioblastoma (GBM) and the standard treatment is

surgical resection followed by radiation and chemotherapy, which does not dramatically improve clinical outcome.

Calcium (Ca2+) dependent signalling pathways are pivotal in the homeostasis of brain tissue. A key element of the cellular response to Ca2+ signals is the action of calcineurin (CN), a Ca2+-

and calmodulin-dependent phosphatase firstly discovered in brain tissue, where is highly abundant1 and described later in many other tissues (reviewed in2,3). CN activation regulates the

activation of the Nuclear Factor of Activated T cells (NFAT) family of transcription factors4,5.

NFAT family includes four classic members: c1, c2, c3 and c4 which were first described in immune cells4,5 and have been associated with malignancies and tumour progression (reviewed in6,7).

NFATc members expression has been shown in astrocytoma8, C69 and U251 glioma cell lines10. Inhibition of the Ca/CN/NFATc pathway by cyclosporine A (CsA) and FK506 has a negative impact on

the growth/survival rate of rat and human GBM cells and on glioma invasion capacity11,12. Accordingly, CsA infusion inhibits tumour growth of implanted glioma in vivo11. Although the

relevance of the Ca/CN/NFATc pathway in glioma biology has been already described by using pharmacological intervention, the mechanism and contribution of the different individual NFATc

members, and their transcriptional program, remain to be fully assessed.

Cytokines have been proven to be potent mediators of antitumor immunity, and overall, their use as treatment for glioma has produced mixed results. The initial preclinical successes using

systemic or local delivery of recombinant cytokines failed to adequately translate into clinical benefit13, but the search for possible cytokine or other immune based therapies remains to be

fully explored. Cytokines and cytokine receptors expressed by glioma cells have been shown to modulate cell invasiveness, survival, proliferation and angiogenesis14, and many of these

cytokines are over-expressed in high grade human glioma15. Cytokines and chemokines prevalently produced in glioma tissue include Tumour Necrosis Factor alpha (TNF-α)16, IL-8

(CXCL8)17,18,19, macrophage chemo attractant protein-1 MCP-1/CCL220, granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF2)21 and Chemokine Receptor 3 (CXCR3). In other cellular

systems, the expression of these cytokines has demonstrated to be sensitive to the inhibitors of the Ca/CN signalling pathway such as CsA22,23,24,25. The source of these molecules,

attributable to glioma cell, surrounding glial cells, tumour-associated macrophages (TAMS), neuronal or lymphocytes, remains to be fully elucidated.

In this work, we evaluated the expression of the individual NFATc members and investigated the role of the most abundant, NFATc3, by exogenous activation and knock-down strategies. We showed

that NFATc3 has a positive role in tumour growth, since NFATc3 knock-down inhibits both proliferation and migration of U251 glioma cells as a model. Our results suggest that NFATc3 alone is

able to modulate a cytokine network required for the normal growth of glioma. Tools limiting NFATc3 might be considered useful to explore future therapeutic approaches against glioma.

CN/NFATc pathway has been previously implicated in the regulation of glioma growth by using pharmacological inhibitors9,26. Nevertheless, the expression and the contribution of the different

NFATc members in glioma cells have not been completely assessed.

U251 is a cell line widely established as a model for human glioma of astrocytic nature27. First, we evaluated the expression of the individual NFATc members (c1 to c4) by quantitative PCR

(qPCR) in U251 cells, together with a collection of human glioblastoma (hGB) cell lines obtained from xenografts. We found that NFATc3 has the highest expression levels in both hGB cell

lines and U251. NFATc1 is also clearly expressed, but in relative less amount. NFATc2 transcript was observed only in some hGB cell lines but hardly detected in U251, and NFATc4 expression

varied among hGB cell lines (Fig. 1A), while it was detectable only in starved U251 (data not shown). In summary, U251 cells have a NFATc expression pattern similar to most of the primary

hGB tested, and NFATc3 and NFATc1 are consistently expressed in both models.

Analysis of NFATc expression and calcium/Calcineurin/NFAT signalling in glioma cells. (A) NFATc1, NFATc2, NFATc3, and NFATc4 mRNAs from U251 and different human Glioblastoma lines (hGB) from

xenografts were amplified by TaqMan RT-PCR. NFATc mRNA was normalized to the expression of TBP as endogenous gene. Results are shown as dCt (Ct NFATc − Ct TBP). (B) Representative

immunoblot showing endogenous expression of NFATc3 and NFATc1 in U251 total protein lysates. Cells were 1 hour pre-treated with 200 ng/mL CsA (lanes 2, 5 and 6) and then, non-stimulated (ns)

as control or stimulated for 30 minutes with 1 μM ionophore alone (Io) or in combination with 20 ng/mL PMA (PIo). (C) Representative immunoblot showing endogenous RCAN1-4 protein

expression. β-actin expression was used as loading control. Glioma total protein lysates from U251 or hGB were pre-treated without (lanes 1 to 3) or with CsA (200 ng/mL) (lanes 4 to 6) and

then stimulated for 4 hours with Io (1 μM) or in combination with PMA (20 ng/mLPIo as indicated. (D) In the upper panel, RCAN1-4 mRNA was amplified from total RNA by TaqMan RT-PCR. U251 were

exposed 4 h to vehicle, Io (1 μM) or thapsigargin A (Tp, 10 nM). RCAN1-4 mRNA was quantified in arbitrary units normalized to the expression of human TBP. Representative experiments of a

minimum of three are shown; values are the mean ± SD of triplicate RT-PCR determinations for each condition. ***P