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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.

Altres ajuts: acords transformatius de la UAB 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.

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., The work was partially supported by funds from the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO) (BIO2016‐78006R, to MP; BFU2015‐71092‐P to IF; SAF2015‐66365 and RTC‐2015‐3386‐1, to MP‐R; Units of Excellence María de Maeztu awards to IF (MDM‐2014‐0435) and to JCP (MDM‐2017–076). Some of the funds were cofinanced with the European Fund for Regional Development (FEDER). We acknowledge additional support from Generalitat de Catalunya (2014‐SGR‐541 and 2017‐SGR‐191). A.B. was funded by the RTC‐2015‐3381‐1 grant. S.C. was a recipient of a short‐term fellowship from the Iranian Government.

The tumor suppressor p21 regulates cell cycle progression and peaks at mid/late G1. However, the mechanisms regulating its expression during cell cycle are poorly understood. We found that embryonic fibroblasts from p27 null mice at early passages progress slowly through the cell cycle. These cells present an elevated basal expression of p21 suggesting that p27 participates to its repression. Mechanistically, we found that p27 represses the expression of Pitx2 (an activator of p21 expression) by associating with the ASE-regulatory region of this gene together with an E2F4 repressive complex. Furthermore, we found that Pitx2 binds to the p21 promoter and induces its transcription. Finally, silencing Pitx2 or p21 in proliferating cells accelerates DNA replication and cell cycle progression. Collectively, these results demonstrate an unprecedented connection between p27, Pitx2 and p21 relevant for the regulation of cell cycle progression and cancer and for understanding human pathologies associated with p27 germline mutations.

The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.

The aim of this study was to investigate the expression of platelet-derived growth factor (PDGF) ligands A and B and receptors α and β in cerebral arteriovenous and cavernous malformations. Fifteen arteriovenous malformation (AVM) and 15 cerebral cavernous malformation (CCM) tissue samples were immunostained for PDGF ligands A and B, PDGF receptors (PDGFR) α and β, and vascular endothelial growth factor. Tissues were compared in terms of expression levels within various vascular layers, and the results were confirmed using western blotting. AVM had higher levels of PDGF-A expression than CCM (p = 0.004, 0.009, 0.001, and 0.027, for endothelium, media, adventitia, and perilesional tissue, respectively) and western blotting showed that there was higher expression of PDGFR-α in AVM tissues. In contrast, CCM endothelium, media, and adventitia had higher PDGF-B expression compared with AVM (p = 0.007, 0.001, and 0.039, respectively). PDGFR-β expression was also significantly higher in the endothelium of CCM tissue (p = 0.007). Overexpression of PDGF ligands and receptors in AVM and CCM may mean that therapeutic strategies targeting the PDGF pathway could be useful in the treatment of these two malformations.

Background: Dose- and time-dependent effects of doxorubicin on telomerase activity (TA) and expression levels of hTERT, Bcl-2, Bcl-xL and Bax were investigated in doxorubicin-resistant and -sensitive MCF-7 cells. Methods: Doxorubicin-resistant MCF-7/R was developed from sensitive MCF-7 breast carcinoma cell line and acquired resistance was demonstrated by XTT and mRNA analysis of MDR1 and MRP1 genes. Expression levels were determined by RT-PCR. Newly developed rapid and simple TRAP-silver staining assay was used to assess TA levels. Results: Doxorubicin-selected MCF-7 cells were 107-fold resistant to the drug and overexpress MDR1 and MRP1 genes. 72 h doxorubicin incubation caused a decrease in TA in parallel with a small decrease in hTERT level in both sensitive and resistant cells. Bcl-2 expression level decreased upon doxorubicin application in sensitive cells. However, the Bcl-xLlevel increased in sensitive cells after 72 h of doxorubicin incubation. Conclusion: This report demonstrates the inhibitory effects of doxorubicin on TA in both resistant and sensitive MCF-7 cells possibly through modulation of the apoptotic pathway genes.

BACKGROUND The primary treatment for craniopharyngiomas is total excision, but recurrence is common. However, current knowledge on the mechanisms of recurrence is limited. OBJECTIVE We hypothesized that recurrence is linked to the angiogenesis of the tumor. Recurrent and nonrecurrent tumor samples were compared with regard to expression of angiogenesis-related factors and angiogenic capacity in a corneal angiogenesis model. METHODS Specimens of 4 recurrent and 6 nonrecurrent tumors were selected from 57 patients with adamantinomatous craniopharyngiomas. Sections were immunohistochemically stained with antibodies for vascular endothelial growth factor (VEGF), fibronectin, fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-A, PDGF-B, platelet-derived growth factor receptor (PDGFR)-alpha, and PDGFR-beta. Expression levels were graded using a 4-point scoring system and were compared. For corneal angiogenesis assay, tissue samples were inoculated in a micropocket created on the rat eye, and microvessels were counted on days 3, 5, 7, and 9 to evaluate angiogenic potential. RESULTS Expression of PDGFR-alpha and FGF-2 were significantly higher for recurrent tumors (P = .02 and P = .01). However, recurrent and nonrecurrent tumors did not differ in the expressions of other ligands and receptors (PDGF-A, PDGF-B, and PDGFR-beta). Recurrent tumors displayed a higher angiogenic potential starting from the fifth day of corneal angiogenesis assay. CONCLUSION These findings suggest a relationship between recurrence of craniopharyngiomas and angiogenesis. New treatment modalities with selective PDGFR-alpha blockers may represent a novel and effective therapeutic option for the treatment of craniopharyngiomas.

The treatment of glomus jugulare tumors represents a challenge for the neurosurgeon, since they invade major vessels and compress critical cranial nerves, resulting in significant morbidity from tumor resection. Among alternative and complementary treatment options, gamma knife radiosurgery is a less invasive procedure and may provide better protection of vital structures. This study aimed to evaluate the efficacy and long-term outcomes of gamma knife surgery in the treatment of these tumors in a large series with the longest follow-up period compared with previous reports. A total of 18 patients with glomus jugulare tumors that underwent gamma knife radiosurgery (GKS) were included. Eleven patients had a history of previous microsurgical treatment. The mean marginal radiation dose was 15.6 Gy (median 15 Gy, range 13-20 Gy). Patients were followed for a mean period of 52.7 months (median 41.5 months); the effect of gamma knife radiosurgery was evaluated using magnetic resonance (MR) images. Based on the last MR images, tumor control could be achieved in 17 out of 18 patients (94.4%). No complications such as radiation-induced peritumoral edema or radiation necrosis occurred. Neurological follow-up examinations revealed improved clinical status in ten patients (55.6%), stable neurological status in seven (38.9%), and deterioration in one patient (5.5%). At the last visit, 17 out of 18 patients were alive. Our results indicate that stereotactic radiosurgery is an effective and safe treatment modality in the management of glomus jugulare tumors, particularly for residual or previously untreated small tumors.

This study aimed to compare cerebral arteriovenous malformations (AVM) and cerebral cavernous malformations (CCM) with regard to the immunohistochemical expressions of matrix metalloproteinases (MMP) and selected extracellular matrix (ECM) proteins, which have a role in the regulation of angiogenesis. Fresh-frozen surgical specimens from patients with AVM (n = 14) and CCM (n = 15) were immunohistochemically stained with antibodies for MMP-2, MMP-9, laminin, fibronectin and tenascin. To compare AVM and CCM, expression of each protein was graded using a four-point scoring system for each histological layer of the lesion. MMP-2 and MMP-9 were more strongly expressed in the vascular walls of CCMs compared to AVMs for all comparable layers: endothelium, subendothelium and the perivascular space. The stronger expression of MMP and other ECM proteins associated with early angiogenesis in CCMs compared to AVMs may support the hypothesis that CCMs occur at an earlier embryogenic stage than AVMs.

Dose- and time-dependent effects of doxorubicin on telomerase activity (TA) and expression levels of hTERT, Bcl-2, Bcl-x(L) and Bax were investigated in doxorubicin-resistant and -sensitive MCF-7 cells.Doxorubicin-resistant MCF-7/R was developed from sensitive MCF-7 breast carcinoma cell line and acquired resistance was demonstrated by XTT and mRNA analysis of MDR1 and MRP1 genes. Expression levels were determined by RT-PCR. Newly developed rapid and simple TRAP-silver staining assay was used to assess TA levels.Doxorubicin-selected MCF-7 cells were 107-fold resistant to the drug and overexpress MDR1 and MRP1 genes. 72 h doxorubicin incubation caused a decrease in TA in parallel with a small decrease in hTERT level in both sensitive and resistant cells. Bcl-2 expression level decreased upon doxorubicin application in sensitive cells. However, the Bcl-x(L)level increased in sensitive cells after 72 h of doxorubicin incubation.This report demonstrates the inhibitory effects of doxorubicin on TA in both resistant and sensitive MCF-7 cells possibly through modulation of the apoptotic pathway genes.

Background The current knowledge on molecular pathogenesis of cerebral vascular malformations (CVM), which are believed to arise during development, is very limited. To unravel the molecular mechanisms involved in CVMs, a detailed understanding of the brain vascular development at molecular level is crucial. In this study, we aimed to explore the temporal and comparative expression profile of angiogenesis-related genes in the establishment of brain vasculature. Methods Expression of a total of 113 angiogenesis-related genes during murine brain development has been analyzed using low-density array systems designed for angiogenesis-related genes. Bai1 (brain specific angiogenesis inhibitor-1), a recently identified novel anti-angiogenic gene, has been selected for further characterization. Results We found that 62 out of 113 analyzed genes have expression in brain development at varying levels. Nineteen of these were differentially expressed between embryonic and postnatal stages (>1.5 fold). Bai1 is strongly expressed on growing blood vessels of cerebral cortex and hippocampus, partially expressed in the lateral regions of striatum, but mostly absent on the thalamus. Conclusion By showing the comparative expression analysis of angiogenesis-related genes throughout brain development, the data presented here will be a crucial addition to further functional studies on cerebrovascular research.