Control of the differentiation of regulatory T cells and T(H)17 cells by the DNA-binding inhibitor Id3.

The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4(+) T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells). We identify two transforming growth factor-ß1 (TGF-ß1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3(-/-) CD4(+) T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3(-/-) T cells showed greater differentiation into the T(H)17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-ß-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors.

Activating mutations in GNAQ and GNA11, encoding members of the Gα(q) family of G protein α subunits, are the driver oncogenes in uveal melanoma, and mutations in Gq-linked G protein-coupled receptors have been identified recently in numerous human malignancies. How Gα(q) and its coupled receptors transduce mitogenic signals is still unclear because of the complexity of signaling events perturbed upon Gq activation. Using a synthetic-biology approach and a genome-wide RNAi screen, we found that a highly conserved guanine nucleotide exchange factor, Trio, is essential for activating Rho- and Rac-regulated signaling pathways acting on JNK and p38, and thereby transducing proliferative signals from Gα(q) to the nucleus independently of phospholipase C-ß. Indeed, whereas many biological responses elicited by Gq depend on the transient activation of second-messenger systems, Gq utilizes a hard-wired protein-protein-interaction-based signaling circuitry to achieve the sustained stimulation of proliferative pathways, thereby controlling normal and aberrant cell growth.

PLCG1 mutations in cutaneous T-cell lymphomas.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of primary cutaneous T-cell lymphoproliferative processes, mainly composed of mycosis fungoides and Sézary syndrome, the aggressive forms of which lack an effective treatment. The molecular pathogenesis of CTCL is largely unknown, although neoplastic cells show increased signaling from T-cell receptors (TCRs). DNAs from 11 patients with CTCL, both normal and tumoral, were target-enriched and sequenced by massive parallel sequencing for a selection of 524 TCR-signaling-related genes. Identified variants were validated by capillary sequencing. Multiple mutations were found that affected several signaling pathways, such as TCRs, nuclear factor κB, or Janus kinase/signal transducer and activator of transcription, but PLCG1 was found to be mutated in 3 samples, 2 of which featured a redundant mutation (c.1034T>C, S345F) in exon 11 that affects the PLCx protein catalytic domain. This mutation was further analyzed by quantitative polymerase chain reaction genotyping in a new cohort of 42 patients with CTCL, where it was found in 19% of samples. Immunohistochemical analysis for nuclear factor of activated T cells (NFAT) showed that PLCG1-mutated cases exhibited strong NFAT nuclear immunostaining. Functional studies demonstrated that PLCG1 mutants elicited increased downstream signaling toward NFAT activation, and inhibition of this pathway resulted in reduced CTCL cell proliferation and cell viability. Thus, increased proliferative and survival mechanisms in CTCL may partially depend on the acquisition of somatic mutations in PLCG1 and other genes that are essential for normal T-cell differentiation.

B-cell lymphoma mutations: improving diagnostics and enabling targeted therapies.

B-cell lymphomas comprise an increasing number of clinicopathological entities whose characterization has historically been based mainly on histopathological features. In recent decades, the analysis of chromosomal aberrations as well as gene and miRNA expression profile studies have helped distinguish particular tumor types and also enabled the detection of a number of targets with therapeutic implications, such as those activated downstream of the B-cell receptor. Our ability to identify the mechanisms involved in B-cell lymphoma pathogenesis has been boosted recently through the use of Next Generation Sequencing techniques in the analysis of human cancer. This work summarizes the recent findings in the molecular pathogenesis of B-cell neoplasms with special focus on those clinically relevant somatic mutations with the potential to be explored as candidates for the development of new targeted therapies. Our work includes a comparison between the mutational indexes and ranges observed in B-cell lymphomas and also with other solid tumors and describes the most striking mutational data for the major B-cell neoplasms. This review describes a highly dynamic field that currently offers many opportunities for personalized therapy, although there is still much to be gained from the further molecular characterization of these clinicopathological entities.

[The role of estrogen receptor alpha in breast cancer cell proliferation mediated by progestins]. / El receptor de estrógenos alfa como mediador del efecto proliferativo de progestágenos en cáncer de mama.

In C4-HD murine mammary carcinomas and in human breast cancer T47D cells, we showed that medroxyprogesterone acetate (MPA) induces a nuclear physical association between estrogen receptor alpha (ERa) and progesterone receptors (PR). The blockade of ERa inhibits cell proliferation mediated by progestins. We hypothesized that this nuclear association between ERa/PR is necessary to trigger progestin-induced cell proliferation and tumor growth. We demonstrated that fulvestrant (FUL, ICI182.780) induced complete regression of C4-HD tumors growing with progestins. MPA treatment induced an early increase in both CCND1 and MYC expression in T47D cells. The blockade of ERa prevented the MPA-dependent transcription of both genes. Specific binding of PR/ERa was observed at the same MPA-sensitive regions at the CCND1 and MYC gene promoters after chromatin immunoprecipitation (ChIP) analysis. ICI inhibited binding of ERa to both gene regulatory sequences while PR binding was unaffected. The nuclear colocalization between both receptors in T47D cells was confirmed by: confocal microscopy, Duolink assays and co-immunoprecipitation assays. In breast cancer samples we also observed a nuclear interaction between both steroid receptors. Our results indicate that the presence of ERa interacting with activated PR at the CCND1 and MYC promoters is required to trigger progestin-induced gene transcription and cell proliferation in breast cancer cells.

c-Myc inhibits Ras-mediated differentiation of pheochromocytoma cells by blocking c-Jun up-regulation.

Although mutant Ras proteins were originally described as transforming oncoproteins, they induce growth arrest, senescence, and/or differentiation in many cell types. c-Myc is an oncogenic transcription factor that cooperates with Ras in cellular transformation and oncogenesis. However, the Myc-Ras relationship in cellular differentiation is largely unknown. Here, we have analyzed the effects of c-Myc on PC12-derived cells (UR61 cell line), harboring an inducible N-Ras oncogene. In these cells, Ras activation induces neuronal-like differentiation by a process involving c-Jun activation. We found that c-Myc inhibited Ras-mediated differentiation by a mechanism that involves the blockade of c-Jun induction in response to Ras signal. Accordingly, ectopically expressed c-Jun could bypass c-Myc impediment of Ras-induced differentiation and activator protein 1 activation. Interestingly, it did not rescue the proliferative arrest elicited by Ras and did not enhance the differentiation-associated apoptosis. The blockade of Ras-mediated induction of c-Jun takes place at the level of c-Jun proximal promoter. Mutational analysis revealed that c-Myc regions involved in DNA binding and transactivation are required to block differentiation and c-Jun induction. c-Myc does not seem to require Miz-1 to inhibit differentiation and block c-Jun induction. Furthermore, Max is not required for c-Myc activity, as UR61 cells lack a functional Max gene. c-Myc-inhibitory effect on the Ras/c-Jun connection is not restricted to UR61 cells as it can occur in other cell types as K562 or HEK293. In conclusion, we describe a novel interplay between c-Myc and c-Jun that controls the ability of Ras to trigger the differentiation program of pheochromocytoma cells.

Nuclear compartmentalization and dynamics of the poly(A)-binding protein nuclear 1 (PABPN1) inclusions in supraoptic neurons under physiological and osmotic stress conditions.

Nuclear aggregation of the expanded polyalanine tract in the poly(A)-binding protein nuclear 1 (PABPN1) is the pathological hallmark of oculopharyngeal muscular dystrophy. However, wild type PABPN1 aggregates into nuclear inclusion in oxytocin-producing neurons under physiological conditions. In this study we have analyzed the nuclear organization and dynamics of PABPN1 inclusions in oxytocin-producing neurons. We demonstrated that PABPN1 inclusions represent a distinct compartment of the interchromatin region. They establish a spatial relationship with nuclear speckles, Cajal bodies and clastosomes. PABPN1 inclusions accumulate poly(A) RNA, but do not concentrate highly expressed mRNAs in oxytocin producing neurons and the mRNA-binding proteins hnRNP C, Y14 and REF. PABPN1 inclusions are dynamic structures that appear during the postnatal period and their number decrease in response to the activation of transcription. Our results support that the RNA retained in the PABPN1 inclusions is a noncoding regulatory RNA involved in some aspects of nuclear RNA metabolism.

Novel triiodophenol derivatives induce caspase-independent mitochondrial cell death in leukemia cells inhibited by Myc.

2,4,6-Triiodophenol (Bobel-24, AM-24) was originally described as a nonsteroid antiinflammatory molecule. We have synthesized three derivatives of Bobel-24 (Bobel-4, Bobel-16, and Bobel-30) and tested their activities as putative antileukemic agents. We have found that Bobel-24 and Bobel-16 were dual inhibitors of cyclooxygenase and 5-lipoxygenase, whereas Bobel-4 and Bobel-30 were selective against 5-lipoxygenase. We have tested the antiproliferative activity of these compounds on a panel of cell lines derived from myeloid and lymphoid leukemias (K562, Raji, HL-60, and Molt4). The cytotoxic IC(50) in these cell lines ranged between 14 and 50 micromol/L, but it was higher for nontransformed cells such as 32D, NIH3T3, or human leukocytes. All compounds showed cytotoxic activity on all tested cell lines, accompanied by DNA synthesis inhibition and arrest in the G(0)/G(1) phase. Bobel-16, Bobel-4, and Bobel-24 induced a caspase-independent cell death in K562 and Raji cells, accompanied by chromatin condensation, cytochrome c release, and dissipation of mitochondrial membrane potential in a concentration-dependent manner and production of reactive oxygen species. As the proto-oncogene MYC is involved in mitochondrial biogenesis and survival of leukemia cells, we tested its effect on bobel activity. Bobel-24 induced down-regulation of MYC in K562 and, consistently, ectopic expression of MYC results in partial protection towards the cytotoxic effect of Bobel-24. In conclusion, Bobel derivatives induce a caspase- and Bcl-2-independent cell death in which mitochondrial permeabilization and MYC down-regulation are involved. Bobels may serve as prototypes for the development of new agents for the therapy of leukemia.

Colorectal adenomas contain multiple somatic mutations that do not coincide with synchronous adenocarcinoma specimens.

We have performed a comparative ultrasequencing study of multiple colorectal lesions obtained simultaneously from four patients. Our data show that benign lesions (adenomatous or hyperplastic polyps) contain a high mutational load. Additionally multiple synchronous colorectal lesions show non overlapping mutational signatures highlighting the degree of heterogeneity between multiple specimens in the same patient. Observations in these cases imply that considering not only the number of mutations but an effective oncogenic combination of mutations can determine the malignant progression of colorectal lesions.

Individualized strategies to target specific mechanisms of disease in malignant melanoma patients displaying unique mutational signatures.

Targeted treatment of advanced melanoma could benefit from the precise molecular characterization of melanoma samples. Using a melanoma-specific selection of 217 genes, we performed targeted deep sequencing of a series of biopsies, from advanced melanoma cases, with a Breslow index of ≥ 4 mm, and/or with a loco-regional infiltration in lymph nodes or presenting distant metastasis, as well of a collection of human cell lines. This approach detected 3-4 mutations per case, constituting unique mutational signatures associated with specific inhibitor sensitivity. Functionally, case-specific combinations of inhibitors that simultaneously targeted MAPK-dependent and MAPK-independent mechanisms were most effective at inhibiting melanoma growth, against each specific mutational background. These observations were challenged by characterizing a freshly resected biopsy from a metastatic lesion located in the skin and soft tissue and by testing its associated therapy ex vivo and in vivo using melanocytes and patient-derived xenografted mice, respectively. The results show that upon mutational characterization of advanced melanoma patients, specific mutational profiles can be used for selecting drugs that simultaneously target several deregulated genes/pathways involved in tumor generation or progression.

Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry.

Mutually exclusive activating mutations in the GNAQ and GNA11 oncogenes, encoding heterotrimeric Gαq family members, have been identified in ∼ 83% and ∼ 6% of uveal and skin melanomas, respectively. However, the molecular events underlying these GNAQ-driven malignancies are not yet defined, thus limiting the ability to develop cancer-targeted therapies. Here, we focused on the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway that controls organ size. We found that Gαq stimulates YAP through a Trio-Rho/Rac signaling circuitry promoting actin polymerization, independently of phospholipase Cß and the canonical Hippo pathway. Furthermore, we show that Gαq promotes the YAP-dependent growth of uveal melanoma cells, thereby identifying YAP as a suitable therapeutic target in uveal melanoma, a GNAQ/GNA11-initiated human malignancy.

Interleukin-21 expression and its association with proinflammatory cytokines in untreated chronic periodontitis patients.

BACKGROUND: Interleukin-21 (IL-21) controls the differentiation of T-helper Th17 cells and induces the production of IL-17 in this T-cell subtype. The aim of this study is to determine the relative expression of IL-21 in gingival tissues of chronic periodontitis patients and correlate/associate this expression with proinflammatory cytokines and clinical parameters of disease. METHODS: Samples of gingival biopsies were collected from chronic periodontitis patients (n = 10) and controls (n = 8). The mRNA expressions of IL-21, IL-1ß, IL-6, IL-17, IL-23, IL-10, and transforming growth factor-ß1 (TGF-ß1) were quantified using real-time reverse transcription-polymerase chain reaction. IL-21 levels were compared between chronic periodontitis and healthy gingival tissues and correlated with cytokine and clinical parameters of tissue destruction. RESULTS: A significant overexpression of IL-21, IL-1ß, IL-6, IL-17, and IL-23p19 was detected in periodontal disease-affected tissues compared to healthy gingival tissues. IL-10 and TGF-ß1 were, however, downregulated in periodontal lesions. IL-21 yielded significant positive correlations with probing depth, clinical attachment level, IL-1ß, and IL-6. In addition, IL-21 was negatively correlated with IL-10 and TGF-ß1. CONCLUSIONS: IL-21 was overexpressed in chronic periodontitis gingival tissues and correlated with clinical parameters of periodontal destruction and with proinflammatory cytokines. Therefore, IL-21 might play a role in the tissue destruction that characterizes chronic periodontal disease.

Estrogen receptor alpha mediates progestin-induced mammary tumor growth by interacting with progesterone receptors at the cyclin D1/MYC promoters.

Synthetic progesterone used in contraception drugs (progestins) can promote breast cancer growth, but the mechanisms involved are unknown. Moreover, it remains unclear whether cytoplasmic interactions between the progesterone receptor (PR) and estrogen receptor alpha (ERα) are required for PR activation. In this study, we used a murine progestin-dependent tumor to investigate the role of ERα in progestin-induced tumor cell proliferation. We found that treatment with the progestin medroxyprogesterone acetate (MPA) induced the expression and activation of ERα, as well as rapid nuclear colocalization of activated ERα with PR. Treatment with the pure antiestrogen fulvestrant to block ERα disrupted the interaction of ERα and PR in vitro and induced the regression of MPA-dependent tumor growth in vivo. ERα blockade also prevented an MPA-induced increase in CYCLIN D1 (CCND1) and MYC expression. Chromatin immunoprecipitation studies showed that MPA triggered binding of ERα and PR to the CCND1 and MYC promoters. Interestingly, blockade or RNAi-mediated silencing of ERα inhibited ERα, but not PR binding to both regulatory sequences, indicating that an interaction between ERα and PR at these sites is necessary for MPA-induced gene expression and cell proliferation. We confirmed that nuclear colocalization of both receptors also occurred in human breast cancer samples. Together, our findings argued that ERα-PR association on target gene promoters is essential for progestin-induced cell proliferation.

Interaction between FGFR-2, STAT5, and progesterone receptors in breast cancer.

Fibroblast growth factor (FGF) receptor 2 (FGFR-2) polymorphisms have been associated with an increase in estrogen receptor and progesterone receptor (PR)-positive breast cancer risk; however, a clear mechanistic association between FGFR-2 and steroid hormone receptors remains elusive. In previous works, we have shown a cross talk between FGF2 and progestins in mouse mammary carcinomas. To investigate the mechanisms underlying these interactions and to validate our findings in a human setting, we have used T47D human breast cancer cells and human cancer tissue samples. We showed that medroxyprogesterone acetate (MPA) and FGF2 induced cell proliferation and activation of ERK, AKT, and STAT5 in T47D and in murine C4-HI cells. Nuclear interaction between PR, FGFR-2, and STAT5 after MPA and FGF2 treatment was also showed by confocal microscopy and immunoprecipitation. This effect was associated with increased transcription of PRE and/or GAS reporter genes, and of PR/STAT5-regulated genes and proteins. Two antiprogestins and the FGFR inhibitor PD173074, specifically blocked the effects induced by FGF2 or MPA respectively. The presence of PR/FGFR-2/STAT5 complexes bound to the PRE probe was corroborated by using NoShift transcription and chromatin immunoprecipitation of the MYC promoter. Additionally, we showed that T47D cells stably transfected with constitutively active FGFR-2 gave rise to invasive carcinomas when transplanted into NOD/SCID mice. Nuclear colocalization between PR and FGFR-2/STAT5 was also observed in human breast cancer tissues. This study represents the first demonstration of a nuclear interaction between FGFR-2 and STAT5, as PR coactivators at the DNA progesterone responsive elements, suggesting that FGFRs are valid therapeutic targets for human breast cancer treatment.

MYC in chronic myeloid leukemia: induction of aberrant DNA synthesis and association with poor response to imatinib.

Untreated chronic myeloid leukemia (CML) progresses from chronic phase to blastic crisis (BC). Increased genomic instability, deregulated proliferation, and loss of differentiation appear associated to BC, but the molecular alterations underlying the progression of CML are poorly characterized. MYC oncogene is frequently deregulated in human cancer, often associated with tumor progression. Genomic instability and induction of aberrant DNA replication are described as effects of MYC. In this report, we studied MYC activities in CML cell lines with conditional MYC expression with and without exposure to imatinib, the front-line drug in CML therapy. In cells with conditional MYC expression, MYC did not rescue the proliferation arrest mediated by imatinib but provoked aberrant DNA synthesis and accumulation of cells with 4C content. We studied MYC mRNA expression in 66 CML patients at different phases of the disease, and we found that MYC expression was higher in CML patients at diagnosis than control bone marrows or in patients responding to imatinib. Further, high MYC levels at diagnosis correlated with a poor response to imatinib. MYC expression did not directly correlate with BCR-ABL levels in patients treated with imatinib. Overall our study suggests that, as in other tumor models, MYC-induced aberrant DNA synthesis in CML cells is consistent with MYC overexpression in untreated CML patients and nonresponding patients and supports a role for MYC in CML progression, possibly through promotion of genomic instability.

Activation of Ras and Rho GTPases and MAP Kinases by G-protein-coupled receptors.

A complex intracellular signaling network mediates the multiple biological activities of G-protein-coupled receptors (GPCRs). Among them, monomeric GTPases and a family of closely related proline-targeted serine-threonine kinases, collectively known as Mitogen-Activated Protein Kinases (MAPKs), appears to play central roles in orchestrating the proliferative responses to multiple mitogens that act on GPCRs. Upon GDP/GTP exchange, monomeric GTPases control the phosphorylation of conserved threonine and tyrosine residues in MAPKs by their immediate upstream kinases, increasing their enzymatic activity and inducing their translocation to the nucleus where they phosphorylate transcription factors, thereby regulating the expression of genes playing a key role in normal and aberrant cell growth. Recently, a number of GPCRs have been engineered to provide exclusive activation by synthetic drug-like compounds while becoming insensitive to endogenous ligands. These engineered receptors, named Receptors Activated Solely by Synthetic Ligands (RASSLs), promise better understanding of GPCRs signaling in vitro and in vivo, thus representing ideal tools to selectively modulate MAPK signaling routes controlling a wide range of biological functions, from proliferation to differentiation, migration, invasion, and cell survival or death by apoptosis.