[Cell Cycle 7:3, 1-7; 1 February 2008]; 2008 Landes Bioscience
The calcineurin/NFAT signaling pathway is unique to verte-
brates and clear genetic evidences show that it plays critical roles in orchestrating the intricate cellular interactions that characterize vertebrate development and morphogenesis. In this setting, the transcriptional regulators of the NFAT family func- tion as molecular integrators of specific calcium signals with other signaling pathways, including MAPkinase, WNT or NOTCH. Deregulation of calcineurin/NFAT signaling and/or abnormal expression of its components have recently been reported in solid tumors of epithelial origin, lymphoma and lymphoid leukemia. Our studies in mouse models of human T-ALL/lymphoma shows that persistent activation of calcineurin/NFAT signaling is pro-oncogenic in vivo and can be efficiently targeted by well- characterized calcineurin inhibitors. We further discuss facts and hypotheses concerning the molecular events that may act upstream and downstream of calcineurin and/or NFAT activation in different type of cancer cells. The Calcineurin/NFAT Signaling Pathway Calcineurin (PP2B) is a calcium-calmodulin-dependent serine/ threonine phosphatase implicated in a number of biological processes (reviewed in refs. 13). In vertebrates, calcineurin is a heterodimer composed of a catalytic subunit A (CnA, 5962kDa) and a regula- tory subunit B (CnB, 19kDa). Three genes encoding the catalytic subunits have been described in vertebrates. CnA and are expressed ubiquitously whereas CnA expression is restricted to testis and brain. 2,4,5 In addition to the phosphatase catalytic domain, CnA contains a CnB-binding domain, a calmodulin-binding domain as well as a carboxy-terminal autoinhibitory domain (Fig. 1). The calcineurin regulatory subunit is encoded by two genes: CnB2, which is specifically expressed in testis and CnB1, which exhibits an ubiquitous expression pattern. In mice, deletion of the CnB1 gene completely impairs calcineurin enzymatic activity in somatic tissues and results in embryonic lethality at day 11 of development due to severe defects in vascular patterning. 6 Under physiological conditions, engagement of cell-surface recep- tors coupled to phospholipase C activation (e.g., the antigen receptor in mature T and B cells) results in the generation of Inositol- (1,4,5)trisphosphate (InsP3) and diacylglycerol (DAG). While DAG activates the RAS/PKC pathway, InsP3 mediates the release of calcium from internal stores, which in turn induces the opening of specific store-operated calcium channels (CRAC). This results in the influx of extracellular calcium and the calcium/calmodulin-dependent activation of calcineurin (Fig. 1; reviewed in ref. 7) and the ensuing dephosphorylation of its substrates, including NFAT (Nuclear Factor of Activated T cell) proteins (Fig. 2). NFAT is a family of 5 members: NFATc1, c2, c3, c4 and NFAT5, the latter being the only family member not regulated by calcineurin. Mouse genetic studies have demonstrated a strong epistatic relationship between calcineurin and NFATs activation and function in many developmental processes (reviewed in refs. 6 and 8 and references therein). Perspective The calcineurin/NFAT signaling pathway A novel therapeutic target in leukemia and solid tumors Hind Medyouf and Jacques Ghysdael* CNRS UMR146 and Institut Curie; Centre Universitaire; Orsay, France Key words: calcineurin, NFAT, leukemia, lymphoma, solid tumors *Correspondence to: Jacques Ghysdael; CNRS UMR146 and Institut Curie; Centre Universitaire; Bat 110; Orsay 91405 France; Tel.: 33.1.69863152; Fax: 33.1.69.07.45.25; Email: Jacques.Ghysdael@curie.u-psud.fr Submitted: 11/05/07; Revised: 11/21/07; Accepted: 11/23/07 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/5357 Figure 1. Mechanism of calcineurin activation. Caleincurin is composed of a catalytic (CnA) and a regulatory (CnB) subunit. In unstimulated cells, calmod- ulin is not associated with calcincurin and the CnA C-terminal autoinhibi- tory domain (AID) interacts with the catalytics cleft and inhibits calcineurin phosphatase activity. Signal-evoked increase in intracellular calcuim results in calcium-dependent binding of calmodulin to calcineurin, thus relieving the inhibitory activity of the AID on the catalytics domain and resulting in the dephosphorylation of calcineurin substrates, including NFAT. www.landesbioscience.com Cell Cycle 297 The calcineurin/NFAT signaling pathway in cancer NFATc1, c2 and c3 are the only members expressed in the lymphoid lineage. In resting lymphocytes, NFAT is located in the cytoplasm as a hyperphosphorylated, inactive form. Under these conditions, NFAT phosphorylation is insured by the combined action of several maintenance kinases, including CK1 and DYRK2 that target specific serine residues in the NFAT conserved regulatory domain. Signaling through calcium/calcineurin results in NFAT proteins dephosphorylation, causing a conformational switch that unmasks their nuclear localization sequence (NLS) and allows their translocation to the nucleus, where they bind to specific DNA response elements to regulate transcription in synergy with a number of other transcriptional regulators. Mice with conditional deletion of the CnB1 gene early in thymocytes development exhibit an 80% decrease in thymic cellularity, perturbed pre-TCR signaling and the complete absence of TCR-mediated positive selection of mature T cells. 9 The importance of calcineurin downstream of TCR signaling in peripheral T cells is underscored by the fact that (1) two effective immunosuppressant used in human medicine, FK506 and cyclosporine A (CsA) are inhibitors of calcineurin; (2) patients with a rare form of hereditary severe combined immunodeficiency (SCID) show a selective defect in calcineurin/ NFAT activation. 10 Targeted deletion of CnB1 in B cells shows an essential role for calcineurin in cell proliferation in response to in vitro anti- IgM stimulation of the antigen receptor (BCR). 11
In contrast, B cell responses in vivo are partially independent of calcineurin, likely due to the inte- gration of BCR signaling with other cell surface receptor signals. This study also showed that self antigen-mediated selection of the B1 cell subset is entirely dependent upon calcineurin. 11 Calcineurin and NFAT Factors in Cancer Calcineurin and/or its downstream NFAT targets have recently been implicated in cancer. 12
Tissue sections from invasive ductal breast carci- noma patients display high expression of NFATc2 and NFAT5 in tumor cells. 13 Studies in breast carcinoma cell lines show that NFAT expres- sion and transcriptional activation are induced downstream of integrin signaling, are indepen- dent of calcineurin activity and promote cell migration and matrigel invasion, suggesting a role for NFATc2/NFAT5 in carcinoma invasive- ness in vivo. 13 Buchholz and colleagues have shown that about 70% of pancreatic carcinoma show high level expression of nuclear NFATc1 as compared to healthy pancreatic tissue. 14 Using human-derived pancreatic carcinomas cell lines, the authors demonstrated that the nuclear local- ization of transcriptionally active NFATc1 is a calcineurin-dependent process since it was inhib- ited by CsA. Treatment with CsA also inhibited in vitro cell cycle progression and anchorage-inde- pendent proliferation of the Panc1 cell line. In addition to their proposed implication in solid tumors, the involvement of calcineurin and NFAT is also suspected in hematologic malignancies. Marafioti et al. surveyed a large panel of Non Hodgkin B-cell and T-cell lymphomas for NFATc1 expression and nuclear localization. 15 Nuclear localization of NFATc1 was found in 70% of the cases of Burkitt lymphoma (BL) and about 30% of the cases of diffuse large B cell lymphoma (DLBCL). Nuclear localiza- tion of NFATc1 or dephosphorylation of both NFATc1 and NFATc2 was also observed in DLBCL patient material 16,17 and in aggressive T cell lymphoma. 17 NFAT activation in cell lines derived from DLBCL and T cell acute lymphoblastic leukemia (T-ALL) is calci- neurin-dependent as it was suppressed in response to CsA or FK506 treatment. 16,17 CsA treatment inhibited cell cycle progression and induced apoptosis in these lines. The in vivo relevance of these observations was obtained in mouse models of human T-ALL/ lymphoma induced either by constitutive activation of the JAK/ STAT or NOTCH1 signaling pathways. In both systems, sustained activation of the calcineurin/NFAT signaling module was observed Figure 2. Schematic view of the calcineurin/NFAT signaling pathway. Engagement by their ligand of cell surface receptors coupled to the activation of phospholipase C or phospholipase C results in the hydrolysis of phophatidylinositol 4,5 bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5 triphosphate (InsP3). InsP3 binds to a receptor in the endoplasmic reticulum (ER) to release calcium ions stored in the ER. Store depletion results in the opening of calcium release-activated channels at the plasma membrane and in the import of extracellular calcium. The resulting increase in intracellular calcium activates calmodulin and a series of calmodulin-dependent enzymes, including the protein phosphatase calcineurin (Cn). Caclium- and calmodulin-activated calcineurin dephosphorylates the 1214 serine residue in NFAT regulatory domain that are constitutively phosphorylated in quiescent cells. Phosphorylated NFAT is cyto- solic and its calcium-mediated dephosphorylation by calcineurin leads to a concerted change in conformation, leading to its nuclear translocation. Nuclear NFAT cooperates with a number of other transcriptional regulators to integrate calcium signaling events with other signaling inputs at the transcriptional level. Cycolsporine A (CsA) and FK506 (Tacrolimus) are unrelated compounds that inhibit calcineurin activity towards its protein substrates following their interactions with spe- cific immunophilins, namely cyclophilin A and FKBP12. 298 Cell Cycle 2008; Vol. 7 Issue 3 The calcineurin/NFAT signaling pathway in cancer in leukemic cells and treatment of diseased mice with either CsA or FK506 resulted in leukemia regression linked to inhibition of tumor cell proliferation, induction of apoptosis and stoechiometric NFAT rephosphorylation. 17 Moreover, transduction of leukemic cells with a calcium-independent, constitutively activated mutant of calcineurin was found to enhance their aggressiveness and to enhance leukemia progression in vivo, consistent with an intrinsic requirement for calcineurin in leukemic cells. It is possible that treatment of leukemic mice with CsA or FK506 also inhibited a function(s) in cells of the tumor microenvironment (e.g., bone marrow stromal cells; blood vessel cells) that could assist leukemic cell proliferation or survival. Taken together, these data provide compelling evidence that calci- neurin activation contributes to the pathogenesis of T-ALL and other aggressive lymphoid malignancies. NFAT Proteins as Potential Mediators of Calcineurin Activity in Cancer Members of the NFAT family are prominent targets of calcineurin which are involved in the regulation of a large number of genes crit- ical for proliferation, growth, migration, differentiation and survival of cells in many lineages (reviewed in ref. 3). Since deregulation of these phenotypic traits is commonly observed in cancer cells, an oncogenic potential for the NFAT proteins has long been suspected but only recently reported. Calcium/calcineurin/NFAT signaling was discovered as an essen- tial pathway acting downstream of the TCR to induce expression of cytokine genes and drive proliferation of initially quiescent T cells. 18,19 Furthermore, the importance of this signaling pathway to cell cycle progression, in particular at the G 1 -to-S transition is well established (reviewed in refs. 20 and 21). In line with this, proteins of the NFAT family have been shown to directly regulate positively or negatively the expression of genes implicated in cell cycle control, including CDK4, 22 cyclin A2 23 and p21 (WAF/Cip1). 24 Moreover NFAT loss-of-function or gain-of-function leads to deregulation of D-type and E-type cyclins expression in several cell types, including lymphocytes. 11,25,26 The calcineurin/NFAT signaling module is also involved in cell survival as exemplified by the increased apop- tosis observed in developing DP thymocytes of NFATc3-deficient mice, 27,28 but the molecular mechanisms involved are still debated. Importantly, although acting downstream from well characterized pro-survival factors such as neurotrophins in neuronal cells, the calcineurin/NFAT pathway is not involved in survival of these cells, indicating clear differences depending upon the cellular context. 29
During normal development, the calcineurin/NFAT signaling module plays a critical role in vasculogenesis and angiogenesis and in the regulation of the VEGF pathway 30-32 and its abnormal deregula- tion in cancer cells could be involved in tumor neo-angiogenesis. Available evidence obtained from the different models studied so far indicates that constitutive activation of NFAT through calcineurin-dependent or independent pathways clearly contrib- utes to the expression of one or more of the phenotypic traits that characterize in vitro transformed- and tumor cells. First, enforced expression in the 3T3L1 pre-adipocyte cell line of a constitutively nuclear and transcriptionally active NFATc1 mutant (caNFATc1) obtained by substitution of the phospshorylation/dephosphoryla- tion sites in NFATc1 regulatory domain by alanine, was sufficient to impair terminal differentiation into adipocytes and to induce cellular transformation. 26 The bypass of the G 1 cell cycle checkpoint and long-term proliferation induced by caNFATc1 under reduced serum conditions was associated with upregulation of c-MYC, cyclin D2 and cyclin D3 expression, but whether this reflects direct transcrip- tional deregulation of these genes by NFATc1 was not determined. Enforced expression of caNFATc1 also protected cells from apoptosis normally induced in 3T3-L1 cells in response to complete serum withdrawal, an effect that was associated with the production of autocrine/paracrine survival factors by transformed cells. 26 In cell lines derived from pancreatic adenocarcinoma, CsA treatment and siRNA-mediated knockdown of NFATc1 inhibited their prolif- eration and anchorage-independent growth. 14 Cell lines that resisted CsA treatment were derived from tumors harboring an amplification of the c-MYC protooncogene whereas NFATc1-dependent regula- tion of c-MYC was observed in responsive cells. An NFATc1 binding site was identified in the c-MYC promoter, that overlaps with a previously identified TGF-response element. Promoter studies in transient transfection assays showed NFATc1-mediated activation of a c-MYC promoter construct, suggesting the direct deregula- tion of c-MYC expression by overexpressed NFATc1 in pancreatic cancer. 14 Importantly, enforced expression of c-MYC was found to rescue Panc1 cells proliferation from CsA-mediated calcineurin inhibition, indicating that c-MYC is indeed a downstream target of calcineurin/NFATc1 activation in these cells. In DLBCL-derived cell lines, Ford and colleagues provided evidence that siRNA-medi- ated downregulation of NFATc1 expression results in decreased cell proliferation, impaired cell survival as well as reduced expression of CD154, a ligand of the TNF family that binds CD40. Since DLBCL express CD40, assembly of an autocrine stimulatory mechanism, resulting in the assembly of a CD40 signalososme is proposed to control cell survival and proliferation of DLBCL cell lines. 33 NFAT binding sites were identified in the CD154 promoter and shown to be important for promoter activity in DLBCL cell lines, a property that relied upon NFATc1 acting in synergy with specific members of the NFB family. 16 In the breast carcinoma cell line model, enforced expression of NFATc2 was found to be sufficient to enhance cell invasion in-matrigel assays whereas overexpression of either NFATc2 or NFAT5 induced cell migration. 13 Further studies have shown that NFATc2 overexpression promotes the in vitro invasive pheno- type of breast carcinoma cell lines through the induction of genes such as COX2 (cyclooxygenase 2) and autotaxin in breast carcinoma cells. 34,35 NFATc2 binding sites are found in the promoters of both the autotaxin and COX2 genes, but whether NFATc2 directly deregulates transcription of these genes in breast carcinome cells was not analyzed. In line with their activity as either positive or negative regulators of genes involved in cell survival and proliferation, NFAT proteins have also been described as potential tumor suppressor genes in specific cellular contexts. Ectopic expression of NFATc2 has been reported to promote apoptosis of Burkitt lymphoma derived cell lines, presum- ably through the induction of Nur77, a member of the orphan nuclear receptor superfamily, a transcriptional regulator involved in apoptosis. 36 In addition, NFATc3-deficient mice infected by the murine lymphomagenic retrovirus SL3-3 develop T-cell lymphomas faster and with higher frequencies as compared to wild-type mice or NFATc2-deficient mice, 37 suggesting that NFATc3 could act as a tumor suppressor gene in the T cell lineage. www.landesbioscience.com Cell Cycle 299 The calcineurin/NFAT signaling pathway in cancer Available evidence thus indicates that, depending upon the tumor type considered, an array of deregulated target genes may be acting downstream of activated calcineurin/NFAT to modulate the tumor phenotype. However, a number of questions remain to be answered. First, the in vivo relevance of these observations, mostly made in cell lines, should be addressed in appropriate mouse models of the respec- tive cancers. Second, several NFAT factors are likely to be expressed in tumor cells and the question arises whether the different NFAT play a redundant or specific role in tumorigenesis. For example, clear evidence indicates that different NFATs can have either specific, or redundant or even antagonistic roles in diverse aspects of normal T cell development (reviewed in ref. 8). This complexity also exists in other cell lineages but to what extent its perturbation contributes to tumor development at different phases of tumorigenesis is at present unknown and will likely be distinct in different cancer types. Finally, although mouse genetic studies have demonstrated a strong epistatic relationship between calcineurin and NFATs activa- tion, it is important to note that these transcription factors are not the only calcineurin downstream substrates. Indeed, calcineurin has been shown to positively regulate a number of other targets such as: channels like InsP3R, the ryanodine or the NMDA receptors; 38,39
enzymes like PKA, 40 NO synthase; 41 other transcription factors like MEF2C, 42 MEF2D 43 and the co-activator TORC2; 44 mechanisms involved in mRNA stabilization 45 or miRNA expression. 46 Several of these downstream targets could potentially mediate calcineurin effects in tumor cells. Upstream Signals Leading to Calcineurin/NFAT Activation in Cancer Cells Little information is available concerning the nature of the upstream events that control calcineurin and NFAT activation in tumor cells. Calcineurin-independent upregulation of NFATc2 and NFAT5 expression in mammary tumor cell lines is linked to expres- sion of 4 integrin and the assembly of an 64 heterodimer, but the molecular events downstream of 64 have not been identified. 13 In the TEL-JAK2 and ICN1-induced mouse models we have used, 17 the rapid emergence of leukemia is critically dependent upon pre-TCR signaling and these clonal leukemias express a functional TCR. 47,48
Since the pre-TCR and the TCR are well characterized receptors coupled to calcium/calcineurin/NFAT activation, 9,49 we consid- ered the possibility that sustained calcium/calcineurin signaling in leukemic cells could reflect acquired hypersensitivity of these cells to pre-TCR/TCR-dependent signals. This is not the case since both TEL-JAK2- and ICN1-induced leukemias generated in Rag2-defi- cient micewhich cannot rearrange the genes encoding TCR and and thus cannot express neither the pre-TCR nor a TCRpresent persistent activation of the calcineurin/NFAT signaling module in a similar fashion as leukemias obtained in RAG2-proficient mice (ref. 17 and our unpublished data). In our models of TEL-JAK2 and ICN1-induced T-ALL/lymphoma, continuous activation of the calcineurin/NFAT module was interrupted when leukemic cells were removed from their in vivo environment (e.g., thymus, spleen, lymph nodes) and maintained in tissue culture for short periods of time. 17
This in vitro inactivation is reversible as re-implantation of these cells into syngeneic hosts resulted in reactivation of the calcineurin/NFAT pathway in the transplanted leukemias (our unpublished observa- tions). This suggests that in vivo activation of the calcineurin/NFAT pathway in leukemic cells depends upon autocrine/paracrine signals or signals generated by other cells in the tumor micro-environ- ment. This in vivo/in vitro dichotomy was found in other instances, including human lymphoma samples (our unpublished observa- tions). This also suggests that activation of the calcineurin/NFAT pathway in lymphoid malignancies might be broader than previously anticipated. 15-17 Our experiments also point to the fact that the TEL-JAK2 and ICN1 initiating/maintenance oncoproteins are not sufficient to acti- vate the calcineurin/NFAT module as they remained active under in vitro conditions. 17 This does not exclude that they nevertheless could assist other upstream events in calcineurin/NFAT activation. For example, TEL-JAK2 is known to activate the endogenous AKT protein kinase 50 and persistent activation of AKT could lead to inactivation of GSK3, an export NFAT kinase. Likewise, in kera- tinocytes, ICN1 has been shown to induce the Hes1-dependent repression of the gene encoding calcipressin1 (CSP1), an endogenous inhibitor of calcineurin. 51 Whether this regulation occurs in T-ALL carrying activating mutations in NOTCH1 52 or in other malignan- cies or other cancers remains to be investigated. Cell lines derived from DLBCL and a subset of T-ALL cell lines show activation of the calcineurin/NFAT module, 16,17 suggesting that their establishment selected clones carrying mutations leading to activation of this pathway. Such mutations have in fact been described in lymphoma-derived cell lines. For example, the EL4 mouse T cell lymphoma cell line was found to express a mutant form of calcineurin in which negative regulation of phosphatase activity by the CnA autoinhibitory domain is impaired due to a mutation that replaces an aspartic acid in position 477 by asparagine. 53 In contrast, constitutive activation of calcineurin in the SML B-cell lymphoma cell line was found to result from the expression of a truncated version of the CnA catalytic subunit. 54 Proteolytic activa- tion of caspases appears to be an alternative mode of activation of calcineurin. To date, two different proteases, namely caspase3 and m- calpain, have been shown to induce calcineurin cleavage in a way that enhances its phosphatase activity. 55-57 In Jurkat T cells, activation of caspase3 in response to PHA treatment leads to the cleavage of CnA to generate truncated polypeptides with enhanced catalytic activity. 55
The second protease, m-calpain, activates calcineurin through two distinct mechanisms: (i) the cleavage of CnA that removes its auto- inhibiory domain, (ii) the cleavage of CABIN1, an endogenous inhibitor of calcineurin. However, no cleaved forms of CnA were detected in our T-ALL mouse models in which we have shown that persistent calcineurin activation contributes to the leukemogenic process (our unpublished observations). Mutations in other components of calcium/calmodulin signaling could be involved in the deregulation of calcineurin or other path- ways in tumor cells. Under physiological conditions, calcineurin activity is negatively regulated by a number of endogenous proteins including the calcipressin family (CSP1, CSP2 and CSP3), 58
CABIN1 (CAIN), 59 AKAP79 (KAP5), 60,61 CHP (Calcineurin Homologous Protein) 62 and FKBP38. 63 Therefore, dysregulated expression of these negative regulators could contribute to enhanced calcineurin activation observed in tumor cells. It has been shown that T cells from mouse expressing a truncated version of CABIN1, that is no longer capable of inhibiting calcineurin activity, overexpress a number of cytokine genes (IL2, IL4, IL9, IL13, IFN) due to 300 Cell Cycle 2008; Vol. 7 Issue 3 The calcineurin/NFAT signaling pathway in cancer their hypersensitivity to TCR signals 64 whereas downregulation of DSCR1 favors the anchorage-dependent and anchorage-independent proliferation of a colorectal cancer-derived cell line. 65 Persistent calcineurin activation could also potentially result from deregulated activity of upstream components in calcium signaling. For example, the TRPV6 calcium channel is overexpressed in advanced stages of prostate cancer and studies in the LNCaP prostate cancer cell line show that TRPV6-mediated calcium import is associated with NFAT activation and favors cell survival and proliferation. 66 Calcineurin/NFAT Signaling Pathway as a Potential Target for Therapy The growing body of evidence implicating the activation of the calcineurin/NFAT signaling pathway in progression and/or mainte- nance of solid tumors, lymphoma and leukemia suggests that the use or development of inhibitors of the molecular events acting upstream of calcineurin activation, of calcineurin itself or of critical effectors of calcineurin may be useful in the treatment of these pathologies. CsA and FK506 are structurally unrelated, well characterized immuno- suppressive agents that function as co-drugs after binding to specific endogenous cytoplasmic cyclophilins, namely cyclophilinA (CypA) and FKB12, respectively. Both the CsA-CypA and FK506-FKBP12 complexes physically interact with calcineurin to inhibit its ability to dephosphorylate protein substrates. This property is at the heart of the potent inhibitory properties of CsA and FK506 in the response of T cells to alloantigens and thus of the widespread clinical use of CsA and FK506 to prevent the rejection of organ transplants and in the treatment of aggressive forms of rheumatoid arthritis and psoriasis. 7,69 T-ALL accounts for about 15% of pediatric and 25% of adult cases of ALL. Molecular characterization of T-ALL has identified a number of genes that contribute to cell cycle and growth deregulation (e.g., loss of the CDKN2A locus), impaired differentiation (e.g., deregulated expression of specific Hox genes) and unlimited self-renewal capacity of leukemic cells. Irrespective of their stage of differentiation arrest, approximately 5060% of T-ALL harbor activating mutations in the NOTCH1 gene, 52 implying a central role of deregulated NOTCH signaling in several aspects of T-ALL biology. While 75% of children with T-cell ALL are cured with combination chemotherapy, the remaining 25% suffer either from refractory or relapsed diseases. These figures and the toxic side effects associated with available chemotherapy regimens clearly calls for the search of novel therapeutic options. Our studies in several mouse models of human T-cell leukemia, including the T-ALL/lymphoma induced by activated NOTCH1, show that short-term (714 days) treatment with either CsA or FK506 results in clear anti-leukemic effects and in prolonged survival of treated versus non treated animals. 15,17 These pre-clinical data suggests that the use of these compounds could provide a therapeutic benefit in remission induction or in the consolidation phase of T-ALL treatment and possibly other malignancies that display high calcineurin/NFAT activation. 13,14,16,32 However CsA and FK506 show severe toxic side effects (neurotoxicity, nephrotoxicity, gastrointestinal disturbances, hypertension) and their long term administration (10 years on longer) in transplanted patients is associated with the emergence of specific cancers due to suppression of tumor immunosurveillance mechanisms. These side effects may compromise the usefulness of CsA and FK506 even in short term induction remission protocols. Nevertheless, case reports have described long term remission of specific lymphocytic leukemias after CsA treatment 67 and the additional inhibitory prop- erties of CsA on ABC transporters have been proposed to form the basis of its therapeutic benefit in AML. 68 Interestingly, derivatives of CsA and FK506 have recently been described, including L-732531 (an analog of FK506 69 ) and ISATX247 (an analog of CsA 70,71 ), that show comparable or even higher efficiency toward calcineurin inhibi- tion and reduced renal toxicity as compared to the respective lead compound. 69-71 The alledged limitations linked to direct inhibition of calcineurin activity clearly requires the further dissection of the molecular path- ways involved in its activation in human malignancies. To date, the molecular events leading to persistent calcineurin activation in cancers remain to be identified and, as discussed above are likely to be distinct in different cancer types. Prior studies have clearly demon- strated that under physiological conditions, calcineurin activation is dependent upon the increase in intracellular calcium concentration through a capacitative calcium entry (CCE) via the CRAC chan- nels (reviewed in ref. 7 and references herein). Therefore, it might be expected that inhibition of CCE through specific inhibitors of the CRAC channels would have similar effects as inhibition of calcineurin with CsA and FK506 (reviewed in ref. 7). Two different compounds, namely BTP2 and capsaicin, which fulfill these criteria have recently been described 72-74 and therefore could represent an alternative approach to inhibit calcineurin for the treatment of cancers where persistent calcineurin activation is implicated. These therapeutic options however are likely to suffer from the same limita- tions as those linked to the direct inhibition of calcineurin. It is therefore critical to identify and analyze the respective roles of the downstream effectors involved in the pro-oncogenic activity of calcineurin in different malignancies. As discussed above, available data support the implication of the NFAT family of transcrip- tion factors in cancer and the relevant genes/pathways deregulated by constitutive NFAT activation may represent novel, potential therapeutic targets. However, some of the other previously described calcineurin targets have also been implicated in cancer. For example, the MEF2D transcription factor is a member of the MEF2 family of DNA binding proteins that activate transcription of genes involved in the control of muscle cell differentiation and in the response of neuronal cells and T-lymphocytes to mitogenic and survival signals. In murine retroviral insertional mutagenesis studies, MEF2D has been identified as a candidate oncogene involved in the pathogen- esis of leukemia. 75,76 Thus, deregulation of MEF2 target genes, if it occurs in tumor cells harboring persistent calcineurin activation, may offer alternative targets of therapeutic interest. Acknowledgements HM was supported by fellowships from the Ministre de lEducation Nationale et de la Recherche and lAssociation pour la Recherche contre le Cancer (ARC). Our work was supported by funds from CNRS (Centre National de la Recherche Scientifique), Institut Curie, INCA (Institut National du Cancer), Cancrople Ile-de-France, ANR (Agence Nationale de la Recherche), Ligue Nationale contre le Cancer (quipe Labelise Ligue) and Association for International Cancer Research. www.landesbioscience.com Cell Cycle 301 The calcineurin/NFAT signaling pathway in cancer References 1. Hogan PG, Chen L, Nardone J, Rao A. 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