The PxIxIT motif of the RCAN3 inhibits angiogenesis and tumor progression in Triple Negative breast cancer in immunocompetent mice.

RCAN proteins are endogenous regulators of the calcineurin-cytosolic nuclear factor of activated T cells (CN-NFATc) pathway that bind CN through similar conserved motifs PxIxIT and LxVP of the NFATc family. RCAN1 and RCAN3 protein levels were reported to correlate with overall survival of breast cancer patients. We additionally provided supporting results about RCAN3 role on cancer showing that overexpression of the native PxIxIT sequence of RCAN3-derived R3 peptide (PSVVVH, EGFP-R3178-210) dramatically inhibits tumor growth and tumor angiogenesis in an orthotopic mouse model of Triple Negative Breast Cancer (TNBC) in nude mice. On the other hand, RCAN3 protein and its derived peptide EGFP-R3178-210 bind to CN and inhibit NFAT-mediated cytokine gene expression without affecting CN phosphatase activity suggesting that RCAN3 and EGFP-R3178-210 peptide have tumor suppressor and immunosuppressant activity. Due to the known relationship between tumor development and immune system, as well as the relevance of CN-NFATc in the regulation of the immune system, in the present study we decided to assess the effect of EGFP-R3178-210 peptide in an orthotopic syngeneic TNBC mouse model, in order to ensure that the role of RCAN3 as immunosuppressant do not override its tumor suppressor activity. Our results evidence that EGFP-R3178-210 peptide displays an inhibitory potential on tumor growth and tumor angiogenesis similar to those obtained in the previous orthotopic TNBC model. These results highlight the importance of the RCAN3 peptide as a tumor suppressor protein and totally complement our previous results, indicating that this antitumor activity role is maintained in the presence of a complete functional immune system.

A novel role for an RCAN3-derived peptide as a tumor suppressor in breast cancer.

The members of the human regulators of calcineurin (RCAN) protein family are endogenous regulators of the calcineurin (CN)-cytosolic nuclear factor of activated T-cells (NFATc) pathway activation. This function is explained by the presence of a highly conserved calcipressin inhibitor of calcineurin (CIC) motif in RCAN proteins, which has been shown to compete with NFATc for the binding to CN and therefore are able to inhibit NFATc dephosphorylation and activation by CN. Very recently, emerging roles for NFATc proteins in transformation, tumor angiogenesis and metastasis have been described in different cancer cell types. In this work, we report that the overexpression of RCAN3 dramatically inhibits tumor growth and tumor angiogenesis in an orthotopic human breast cancer model. We suggest that RCAN3 exerts these effects in a CN-dependent manner, as mutation of the CIC motif in RCAN3 abolishes the tumor suppressor effect. Moreover, the expression of the EGFP-R3(178-210) peptide, spanning the CIC motif of RCAN3, is able to reproduce all the antitumor effects of RCAN3 full-length protein. Finally, we show that RCAN3 and the EGFP-R3(178-210) peptide inhibit the CN-NFATc signaling pathway and the induction of the NFATc-dependent gene cyclooxygenase-2. Our work suggests that the EGFP-R3(178-210) peptide possess potent tumor suppressor properties and therefore constitutes a novel lead for the development of potent and specific antitumoral agents. Moreover, we propose the targeting of the CN-NFATc pathway in the tumor cells constitutes an effective way to hamper tumor progression by impairing the paracrine network among tumor, endothelial and polymorphonucleated cells.

RCAN 1 and 3 proteins regulate thymic positive selection.

Cooperation between calcineurin (CN)-NFATc and RAF-MEK-ERK signaling pathways is essential in thymocyte positive selection. It is known that the Regulators of Calcineurin (RCAN) proteins can act either facilitating or suppressing CN-dependent signaling events. Here, we show that RCAN genes are expressed in lymphoid tissues, and address the role of RCAN proteins in T cell development. Overexpression of human RCAN3 and RCAN1 can modulate T cell development by increasing positive selection-related surface markers, as well as the "Erk(hi) competence state" in double positive thymocytes, a characteristic molecular signature of positive selection, without affecting CN activity. We also found that RCAN1/3 interact with RAF kinases and CN in a non-exclusive manner. Our data suggests that the balance of RCAN interactions with CN and/or RAF kinases may influence T cell positive selection.

Protein kinase CK2-dependent phosphorylation of the human Regulators of Calcineurin reveals a novel mechanism regulating the calcineurin-NFATc signaling pathway.

Cyclosporine A and FK506 produce immunosuppression by blocking calcineurin phosphatase activity and consequently activation of cytosolic Nuclear Factor of Activated T-cell (NFATc) transcription factor. Due to the chronic toxicity associated with their administration, the development of more specific immunosuppressants is currently an important unmet medical need. In this context, an immunosuppressant peptide derived from the CIC motif of the human Regulators of Calcineurin (RCAN) proteins has been shown to inhibit NFATc signaling without affecting general phosphatase activity of calcineurin. Here we show that protein kinase CK2 phosphorylates a conserved serine residue within the CIC motif of vertebrate RCANs, which increases its affinity for calcineurin and consequently its inhibition of NFATc-dependent gene expression in activated T-cells. Molecular modeling studies have led us to identify a positively charged interaction site on the surface of calcineurin where the phosphorylated serine residue of the CIC motif would normally locate. Finally, we have also identified RCAN3 as a new phosphoprotein with multiple phosphorylation sites. Therefore, our findings reveal for the first time a novel molecular mechanism underlying the regulation of calcineurin-NFATc signaling by means of phosphorylation of the CIC motif of RCAN proteins. The knowledge of how RCAN proteins modulate the calcineurin-NFATc pathway paves the way for the development of potent novel selective immunosuppressant drugs.

Design and synthesis of a novel non peptide CN-NFATc signaling inhibitor for tumor suppression in triple negative breast cancer.

The Ca2+/calmodulin-mediated phosphatase activity of calcineurin (CN) integrates calcium-mediated signaling with gene expression programs involved in the control of essential cellular processes in health and disease, such as the immune response and the pathogenesis of cancer progression and metastasis. In addition, CN is the target of the immunosuppressive drugs cyclosporine A (CsA) and FK-506 which are the cornerstone of immunosuppressant therapy. Unfortunately, long-term administration of these drugs results in severe side effects. Herein, we describe the design, synthesis and evaluation of new synthetic compounds that are capable of inhibiting NFATc activity in a dose-dependent manner, without interfering on CN phosphatase activity. These compounds were designed using the structure-based pharmacophore model of a peptide-derived PxIxIT sequence binding to calcineurin A subunit. Moreover, these compounds inhibit NFATc-dependent cytokine gene expression, secretion and proliferation of human T CD4+ cells. More importantly, compound 5a reduces tumor weight and shows a tendency to reduce tumor angiogenesis in an orthotopic immunocompetent mouse model of triple negative breast cancer, suggesting that 5a has tumor suppressor activity. These findings validate compound 5a as an agent with therapeutic activity against CN-NFATc and highlight its potential as a tool for drug development with therapeutic purposes.

Intragraft expression of the IL-10 gene is up-regulated in renal protocol biopsies with early interstitial fibrosis, tubular atrophy, and subclinical rejection.

Grafts with subclinical rejection associated with interstitial fibrosis and tubular atrophy (SCR+IF/TA) show poorer survival than grafts with subclinical rejection without IF/TA (SCR). Aiming to detect differences among SCR+IF/TA and SCR, we immunophenotyped the inflammatory infiltrate (CD45, CD3, CD20, CD68) and used a low-density array to determine levels of T(H)1 (interleukin IL-2, IL-3, gamma-interferon, tumor necrosis factor-alpha, lymphotoxin-alpha, lymphotoxin-beta, granulocyte-macrophage colony-stimulating factor) and T(H)2 (IL-4, IL-5, IL-6, IL-10, and IL-13) transcripts as well as of IL-2R (as marker for T-cell activation) in 31 protocol biopsies of renal allografts. Here we show that grafts with early IF/TA and SCR can be distinguished from grafts with SCR on the basis of the activation of IL-10 gene expression and of an increased infiltration by B-lymphocytes in a cellular context in which the degree of T-cell activation is similar in both groups of biopsies, as demonstrated by equivalent levels of IL-2R mRNA. These results suggest that the up-regulation of the IL-10 gene expression, as well as an increased proportion of B-lymphocytes in the inflammatory infiltrates, might be useful as markers of early chronic lesions in grafts with SCR.

A fluorescent polarization-based assay for the identification of disruptors of the RCAN1-calcineurin A protein complex.

Calcineurin is a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programs, including immune response. One of the most studied substrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most commonly used immunosuppressants in transplantation therapies. Unfortunately, their mechanism of action, completely blocking the calcineurin phosphatase activity while also requiring continuous administration, bears severe side effects. During recent years, the family of regulators of calcineurin (RCAN) has been described and studied extensively as modulators of calcineurin signaling pathways. The RCAN1 region, spanning amino acids 198 to 218 and responsible for inhibiting the calcineurin-NFAT signaling pathway in vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin-NFAT interaction without affecting the general calcineurin phosphatase activity. Here we report the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1(198-218)-CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction. Moreover, three small molecules with a potential immunosuppressive effect were also identified.

Seminal plasma microRNAs improve diagnosis/prognosis of prostate cancer in men with moderately altered prostate-specific antigen.

There is an urgent need for accurate non-invasive biomarkers for prostate cancer (PCa) diagnosis and disease risk stratification. Previous data suggests that total seminal plasma (SP) represents a source of miRNAs for screening. We have evaluated a panel of eight PCa-associated miRNAs for their potential use as PCa biomarkers in SP by analyzing their levels using RT-qPCR. Multivariate logistic regression modelling and clinical risk assessment were performed for those SP miRNAs statistically altered between PCa and non-PCa (HCt and/or BPH) groups. Our results provide evidence that altered miRNA expression in PCa tissue can also be detected in total SP. We obtained a clinically useful SP miRNA-based combined model (PSA+miR-142-3p+miR-223-3p+miR-93-5p), which improves PCa specificity of the PSA test, for, firstly, predicting the presence of malignant tumors in a sample from the total population and secondly, and more interestingly for clinicians, for predicting PCa in samples from the positive PSA screening test (PSA>4 ng/ml). Additionally, [PSA+miR-30d-5p+miR-93-5p] and [PSA+miR-30d-5p] models have been shown to be useful for predicting the disease aggressiveness with diagnostic accuracy. In conclusion, our results provide evidence that miRNAs in total SP represent a useful target for evaluation for PCa, which technically simplifies the future use of semen miRNA-based models as non-invasive biomarkers to increase the efficiency of PCa diagnosis and prognosis.

Cis-trans proline isomers in the catalytic domain of calcineurin.

Calcineurin is an essential calcium-activated serine/threonine phosphatase. The six NMR-observable methionine methyl groups in the catalytic domain of human calcineurin Aα (CNA) were assigned and used as reporters of the presence of potential cis-trans isomers in solution. Proline 84 is found in the cis conformation in most calcineurin X-ray structures, and proline 309, which is part of a highly conserved motif in phosphoprotein phosphatases, was modeled with a cis peptide bond in one of the two molecules present in the asymmetric unit of CNA. We mutated each of the two prolines to alanine to force the trans conformation. Solution NMR shows that the P84A CNA mutant exists in two forms, compatible with cis-trans isomers, while the P309A mutant is predominantly in the trans conformation. DATABASE: PDB depositions mentioned PDB 5C1V and 2JOG.

RCAN3, a novel calcineurin inhibitor that down-regulates NFAT-dependent cytokine gene expression.

The regulators of calcineurin (RCAN) proteins, previously known as calcipressins, have been considered to be a well conserved family from yeast to human based on the conservation of their FLISPP motif. Here, after performing a RCAN comparative genomic analysis we propose the existence of a novel functionally closely related RCAN subfamily restricted to vertebrates, the other RCAN proteins being considered only as distantly related members of the family. In addition, while three paralogous RCAN genes are found in vertebrates, there is only one in the other members of Eukarya. Moreover, besides the FLISPP motif, these paralogous genes have two others conserved motifs, the Cn-inhibitor RCAN (CIC) and the PxIxxT, which are restricted to vertebrates. In humans, RCAN1 and RCAN2 bind and inhibit Cn through their C-terminal region. Given the high amino acid identity in this region among human RCANs, authors in the field have hypothesized a role for RCAN3 in inhibiting Cn activity. Here, we demonstrate for the first time that human RCAN3, encoded by the RCAN3 (also known as DSCR1L2) gene, interacts physically and functionally with Cn. This interaction takes place only through the RCAN3 CIC motif. Overexpression of this sequence inhibits Cn activity towards the nuclear factor of activated T cells (NFAT) transcription factors and down-regulates NFAT-dependent cytokine gene expression in activated human Jurkat T cells.

Functional characterization of twelve natural PROS1 mutations associated with anticoagulant protein S deficiency.

BACKGROUND: The molecular mechanisms by which PROS1 mutations result in protein S deficiency are still unknown for many of the mutations, particularly for those that result in a premature termination codon. The aim of this study was to analyze the functional relevance on mRNA and protein expression of 12 natural PROS1 mutations associated with protein S deficiency. DESIGN AND METHODS: Five mutations were nonsense, three were small frameshift deletions, one was c.258,259AG>GT at the 3' end of exon 3, one was p.M640T and the last two were c.-7C>G and p.L15H, found in double heterozygosis as [c.-7C>G;44T>A]. The apparently neutral variant p.R233K was also analyzed. PROS1 cDNA was assessed by reverse transcriptase polymerase chain reaction of platelet mRNA. Expression of mutant proteins was determined by site-directed mutagenesis and analyses of transiently transfected PROS1 mutants in COS-7 cells. RESULTS: Only cDNA from the normal allele was observed from the five nonsense mutations, the frameshift deletion c.1731delT and from c.258,259AG>GT. Both the normal and the mutated alleles were observed from [c.-7C>G;44T>A], c.187,188delTG and p.M640T. Transient expression analyses of PROS1 mutants whose mRNA was normally expressed revealed greatly reduced secretion of p.L15H and c.1272delA, mild secretion values of p.M640T and normal secretion levels of c.-7C>G and, as expected, p.R233K. CONCLUSIONS: Whereas the main cause of quantitative protein S deficiency associated with missense mutations is defective synthesis, stability or secretion of the mutated protein, the main mechanism for the deficiency associated with mutations that generate a premature termination codon is not the synthesis of a truncated protein, but the exclusion of the mutated allele, probably by nonsense-mediated mRNA decay.

Functional characterization of the calcipressin 1 motif that suppresses calcineurin-mediated NFAT-dependent cytokine gene expression in human T cells.

Inhibition of the calcineurin-NFAT signalling pathway is one of the main challenges for immunosuppression therapy to avoid the severe side effects of the current anticalcineurinic drugs, cyclosporin A and FK506. The members of the calcipressin family are endogenous inhibitors of calcineurin. We describe for the first time that two independent motifs within human calcipressin 1, the ELHA and the PxIxxT motifs, interact with calcineurin in an independent functional manner. However, the main finding here is that the ELHA-containing calcineurin-inhibitor CALP1 (CIC) motif is the responsible for the in vivo inhibition of calcineurin-mediated NFAT-dependent cytokine gene expression in human T cells. We believe that the identification of the CIC motif could be used as a starting point for the development of new immunosuppressive drugs for use in transplantation and autoimmune diseases.

Phosphorylation of calcipressin 1 increases its ability to inhibit calcineurin and decreases calcipressin half-life.

Calcipressin 1 is an endogenous inhibitor of calcineurin, which is a serine/threonine phosphatase under the control of Ca(2+) and calmodulin. Calcipressin 1 is encoded by DSCR1, a gene on human chromosome 21 with seven exons, exons 1-4 are alternative first exons (isoforms 1-4). We show that calcipressin 1 isoform 1 has an N-terminal coding region longer than that previously described, and this generates a new polypeptide of 252 amino acids. This polypeptide is able to interact with calcineurin A and to inhibit NF-AT-mediated transcriptional activation. We demonstrate for the first time that endogenous calcipressin 1 exists as a complex together with the calcineurin A and B heterodimer. Calcipressin 1 is a phosphoprotein that increases its capacity to inhibit calcineurin when phosphorylated at the FLISPP motif, and this phosphorylation also controls the half-life of calcipressin 1 by accelerating its degradation. Additionally, we have also detected further phosphorylation sites outside the FLISPP motif and these contribute to the complex phosphorylation pattern of calcipressin 1. Taking all these results into consideration we suggest that phosphorylation of calcipressin 1 is involved in the regulation of the phosphatase activity of calcineurin and can therefore act as a modulator of calcineurin-dependent cellular pathways.

Calcineurin Undergoes a Conformational Switch Evoked via Peptidyl-Prolyl Isomerization.

A limited repertoire of PPP family of serine/threonine phosphatases with a highly conserved catalytic domain acts on thousands of protein targets to orchestrate myriad central biological roles. A major structural reorganization of human calcineurin, a ubiquitous Ser/Thr PPP regulated by calcium and calmodulin and targeted by immunosuppressant drugs cyclosporin A and FK506, is unveiled here. The new conformation involves trans- to cis-isomerization of proline in the SAPNY sequence, highly conserved across PPPs, and remodels the main regulatory site where NFATc transcription factors bind. Transitions between cis- and trans-conformations may involve peptidyl prolyl isomerases such as cyclophilin A and FKBP12, which are known to physically interact with and modulate calcineurin even in the absence of immunosuppressant drugs. Alternative conformations in PPPs provide a new perspective on interactions with substrates and other protein partners and may foster development of more specific inhibitors as drug candidates.

Identification of small-molecule inhibitors of calcineurin-NFATc signaling that mimic the PxIxIT motif of calcineurin binding partners.

Calcineurin (CN), a serine and threonine protein phosphatase that depends on Ca(2+) and calmodulin for its activity, is the target of the immunosuppressant drugs cyclosporin A (CsA) and tacrolimus (FK506). CN dephosphorylates and activates members of the NFATc (nuclear factor of activated T cells) family of transcription factors in T cells by binding to their conserved PxIxIT motif. Upon dephosphorylation, NFATc proteins translocate to the nucleus, where they stimulate the expression of genes encoding cytokines and chemokines that are required for T cell proliferation and the immune response. We performed a pharmacophore-based virtual screening of ~5.5 million commercially available, "drug-like" compounds to identify nonpeptidic compounds that inhibited the CN-dependent activation of NFATc signaling and that could serve as potential drug candidates for immunosuppressive therapy. Of 32 compounds that mimicked the PxIxIT motif, 7 competed with NFATc for binding to CN in vitro without interfering with the phosphatase activity of CN. Furthermore, in activated human CD4(+) T cells, four of the seven compounds inhibited the expression of NFATc-dependent genes, cytokine production, and cell proliferation, suggesting that these may have therapeutic potential as immunosuppressive agents.

Dyrk1A expression pattern supports specific roles of this kinase in the adult central nervous system.

Dyrk1A and its Drosophila orthologue, the protein minibrain (mnb), belong to a family of serine/threonine kinases involved in the development of the central nervous system (CNS). However, additional roles for Dyrk1A have to be proposed, as its expression is still prominent in the adult brain. To gain insight into Dyrk1A physiological roles we have studied the distribution of this kinase in the CNS of mice in adulthood. A homogeneous diffuse immunostaining of variable intensity was detected throughout the neuropile, with the white matter displaying faint Dyrk1A immunoreactivity. Dyrk1A immunostaining was strong in the olfactory bulb, the cerebellar cortex and functionally related structures, the spinal cord and most of the motor nuclei of the midbrain and brain stem. These data agree with a possible implication of this kinase in the physiology of olfaction and motor functions. Cellular and subcellular localisation of Dyrk1A was also studied in primary cell culture of cerebellum, one of the structures showing significant Dyrk1A immunostaining in the adult. The distribution of Dyrk1A in primary cell culture showed the presence of this protein in the nucleus and the cytoplasm of both neurons and astrocytes. Moreover, studies on the subcellular distribution of Dyrk1A in whole brain homogenates of adult mice showed the presence of this protein both in nuclear and cytoplasm-enriched fractions, thus supporting selective functions of this kinase in these two subcellular compartments. The present results showing the distribution of Dyrk1A in widespread areas of the adult CNS and in different subcellular compartments, together with previous reports demonstrating its implication in developmental events concur with the idea of several spatio-temporal functional profiles.

The vertebrate RCAN gene family: novel insights into evolution, structure and regulation.

Recently there has been much interest in the Regulators of Calcineurin (RCAN) proteins which are important endogenous modulators of the calcineurin-NFATc signalling pathway. They have been shown to have a crucial role in cellular programmes such as the immune response, muscle fibre remodelling and memory, but also in pathological processes such as cardiac hypertrophy and neurodegenerative diseases. In vertebrates, the RCAN family form a functional subfamily of three members RCAN1, RCAN2 and RCAN3 whereas only one RCAN is present in the rest of Eukarya. In addition, RCAN genes have been shown to collocate with RUNX and CLIC genes in ACD clusters (ACD21, ACD6 and ACD1). How the RCAN genes and their clustering in ACDs evolved is still unknown. After analysing RCAN gene family evolution using bioinformatic tools, we propose that the three RCAN vertebrate genes within the ACD clusters, which evolved from single copy genes present in invertebrates and lower eukaryotes, are the result of two rounds of whole genome duplication, followed by a segmental duplication. This evolutionary scenario involves the loss or gain of some RCAN genes during evolution. In addition, we have analysed RCAN gene structure and identified the existence of several characteristic features that can be involved in RCAN evolution and gene expression regulation. These included: several transposable elements, CpG islands in the 5' region of the genes, the existence of antisense transcripts (NAT) associated with the three human genes, and considerable evidence for bidirectional promoters that regulate RCAN gene expression. Furthermore, we show that the CpG island associated with the RCAN3 gene promoter is unmethylated and transcriptionally active. All these results provide timely new insights into the molecular mechanisms underlying RCAN function and a more in depth knowledge of this gene family whose members are obvious candidates for the development of future therapies.

Inhibiting the calcineurin-NFAT (nuclear factor of activated T cells) signaling pathway with a regulator of calcineurin-derived peptide without affecting general calcineurin phosphatase activity.

Calcineurin phosphatase plays a crucial role in T cell activation. Dephosphorylation of the nuclear factors of activated T cells (NFATs) by calcineurin is essential for activating cytokine gene expression and, consequently, the immune response. Current immunosuppressive protocols are based mainly on calcineurin inhibitors, cyclosporine A and FK506. Unfortunately, these drugs are associated with severe side effects. Therefore, immunosuppressive agents with higher selectivity and lower toxicity must be identified. The immunosuppressive role of the family of proteins regulators of calcineurin (RCAN, formerly known as DSCR1) which regulate the calcineurin-NFAT signaling pathway, has been described recently. Here, we identify and characterize the minimal RCAN sequence responsible for the inhibition of calcineurin-NFAT signaling in vivo. The RCAN-derived peptide spanning this sequence binds to calcineurin with high affinity. This interaction is competed by a peptide spanning the NFAT PXIXIT sequence, which binds to calcineurin and facilitates NFAT dephosphorylation and activation. Interestingly, the RCAN-derived peptide does not inhibit general calcineurin phosphatase activity, which suggests that it may have a specific immunosuppressive effect on the calcineurin-NFAT signaling pathway. As such, the RCAN-derived peptide could either be considered a highly selective immunosuppressive compound by itself or be used as a new tool for identifying innovative immunosuppressive agents. We developed a low throughput assay, based on the RCAN1-calcineurin interaction, which identifies dipyridamole as an efficient in vivo inhibitor of the calcineurin-NFAT pathway that does not affect calcineurin phosphatase activity.