Thrombin Protease-activated Receptor-1 Signals through Gq- and G13-initiated MAPK Cascades Regulating c-Jun Expression to Induce Cell Transformation

Although the ability of G protein-coupled receptors to stimulate normal and aberrant cell growth has been intensely investigated, the precise nature of the molecular mechanisms underlying their transforming potential are still not fully understood. In this study, we have taken advantage of the potent mitogenic effect of thrombin and the focus-forming activity of one of its receptors, protease-activated receptor-1, to dissect how this receptor coupled to Gi, Gq/11, and G12/13 transduces signals from the membrane to the nucleus to initiate transcriptional events involved in cell transformation. Using endogenous and transfected thrombin receptors in NIH 3T3 cells, ectopic expression of muscarinic receptors coupled to Gq and Gi, and chimeric G protein subunits and murine fibroblasts deficient in Gq/11, and G12/13, we show here that, although coupling to Gi is sufficient to induce ERK activation, the ability to couple to Gq and/or G13 is necessary to induce c-jun expression and cell transformation. Furthermore, we show that Gq and G13 can initiate the activation of MAPK cascades, including JNK, p38, and ERK5, which in turn regulate the activity of transcription factors controlling expression from the c-jun promoter. We also present evidence that c-Jun and the kinases regulating its expression are integral components of the transforming pathway initiated by protease-activated receptor-1

Rac inhibits thrombin-induced Rho activation: evidence of a Pak-dependent GTPase crosstalk

The strict spatio-temporal control of Rho GTPases is critical for many cellular functions, including cell motility, contractility, and growth. In this regard, the prototypical Rho family GTPases, Rho, Rac, and Cdc42 regulate the activity of each other by a still poorly understood mechanism. Indeed, we found that constitutively active forms of Rac inhibit stress fiber formation and Rho stimulation by thrombin. Surprisingly, a mutant of Rac that is unable to activate Pak1 failed to inhibit thrombin signaling to Rho. To explore the underlying mechanism, we investigated whether Pak1 could regulate guanine nucleotide exchange factors (GEFs) for Rho. We found that Pak1 associates with P115-RhoGEF but not with PDZ-RhoGEF or LARG, and knock down experiments revealed that P115-RhoGEF plays a major role in signaling from thrombin receptors to Rho in HEK293T cells. Pak1 binds the DH-PH domain of P115-RhoGEF, thus suggesting a mechanism by which Rac stimulation of Pak1 may disrupt receptor-dependent Rho signaling. In agreement, expression of a dominant-negative Pak-Inhibitory Domain potentiated the activation of Rho by thrombin, and prevented the inhibition of Rho by Rac. These findings indicate that Rac interferes with receptor-dependent Rho stimulation through Pak1, thus providing a mechanism for cross-talk between these two small-GTPases

Unraveling the oral cancer lncRNAome: Identification of novel lncRNAs associated with malignant progression and HPV infection

Objectives The role of long non-coding RNA (lncRNA) expression in human head and neck squamous cell carcinoma (HNSCC) is still poorly understood. In this study, we aimed at establishing the onco-lncRNAome profiling of HNSCC and to identify lncRNAs correlating with prognosis and patient survival. Materials and methods The Atlas of Noncoding RNAs in Cancer (TANRIC) database was employed to retrieve the lncRNA expression information generated from The Cancer Genome Atlas (TCGA) HNSCC RNA-sequencing data. RNA-sequencing data from HNSCC cell lines were also considered for this study. Bioinformatics approaches, such as differential gene expression analysis, survival analysis, principal component analysis, and Co-LncRNA enrichment analysis were performed. Results Using TCGA HNSCC RNA-sequencing data from 426 HNSCC and 42 adjacent normal tissues, we found 728 lncRNA transcripts significantly and differentially expressed in HNSCC. Among the 728 lncRNAs, 55 lncRNAs were significantly associated with poor prognosis, such as overall survival and/or disease-free survival. Next, we found 140 lncRNA transcripts significantly and differentially expressed between Human Papilloma Virus (HPV) positive tumors and HPV negative tumors. Thirty lncRNA transcripts were differentially expressed between TP53 mutated and TP53 wild type tumors. Co-LncRNA analysis suggested that protein-coding genes that are co-expressed with these deregulated lncRNAs might be involved in cancer associated molecular events. With consideration of differential expression of lncRNAs in a HNSCC cell lines panel (n = 22), we found several lncRNAs that may represent potential targets for diagnosis, therapy and prevention of HNSCC. Conclusion LncRNAs profiling could provide novel insights into the potential mechanisms of HNSCC oncogenesis.Centro de Investigaciones Inmunológicas Básicas y Aplicada

A synthetic-lethality RNAi screen reveals an ERK-mTOR co-targeting pro-apoptotic switch in PIK3CA+ oral cancers.

mTOR inhibition has emerged as a promising strategy for head and neck squamous cell carcinomas (HNSCC) treatment. However, most targeted therapies ultimately develop resistance due to the activation of adaptive survival signaling mechanisms limiting the activity of targeted agents. Thus, co-targeting key adaptive mechanisms may enable more effective cancer cell killing. Here, we performed a synthetic lethality screen using shRNA libraries to identify druggable candidates for combinatorial signal inhibition. We found that the ERK pathway was the most highly represented. Combination of rapamycin with trametinib, a MEK1/2 inhibitor, demonstrated strong synergism in HNSCC-derived cells in vitro and in vivo, including HNSCC cells expressing the HRAS and PIK3CA oncogenes. Interestingly, cleaved caspase-3 was potently induced by the combination therapy in PIK3CA+ cells in vitro and tumor xenografts. Moreover, ectopic expression of PIK3CA mutations into PIK3CA- HNSCC cells sensitized them to the pro-apoptotic activity of the combination therapy. These findings indicate that co-targeting the mTOR/ERK pathways may provide a suitable precision strategy for HNSCC treatment. Moreover, PIK3CA+ HNSCC are particularly prone to undergo apoptosis after mTOR and ERK inhibition, thereby providing a potential biomarker of predictive value for the selection of patients that may benefit from this combination therapy

The small GTP-binding proteins Rac1 and Cdc42regulate the activity of the JNK/SAPK signaling pathway

Summaryc-Jun amino-terminal kinases (JNKs) and mitogen-activatedprotein kinases (MAPKs) are closely related; however, they are independently regulated by a variety of environmental stimuli. Although molecules linking growth factor receptors to MAPKs have been recently identified, little is known about pathways controlling JNK activation. Here, we show that in COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Racl and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Racl and Cdc42 in controlling the JNK signaling pathway

VE-cadherin and claudin-5: it takes two to tango

Endothelial barrier function requires the adhesive activity of VE-cadherin and claudin-5, which are key components of adherens and tight endothelial junctions, respectively. Emerging evidence suggests that VE-cadherin controls claudin-5 expression by preventing the nuclear accumulation of FoxO1 and -catenin, which repress the claudin-5 promoter. This indicates that a crosstalk mechanism operates between these junctional structures

Cross-Desensitization and Cointernalization of H1 and H2 Histamine Receptors Reveal New Insights into Histamine Signal Integration

G-protein coupled receptor (GPCR) signaling does not result from sequential activation of a linear pathway of proteins/enzymes, but rather from complex interactions of multiple, branched signaling routes, ie, signaling networks. In this work we present an exhaustive study of the crosstalk between H1 and H2 histamine receptors (H1R and H2R) in U937 cells and CHO transfected cells. By desensitization assays we demonstrated the existence of a cross-desensitization between both receptors independent of protein kinase A (PKA) or C (PKC). H1R agonist stimulation inhibited cell proliferation and induced apoptosis in U937 cells following treatment for 48h. H1R-induced antiproliferative and apoptotic response was inhibited by an H2R agonist suggesting that the crosstalk between both receptors modifies their function. Binding and confocal microscopy studies revealed cointernalization of both receptors upon treatment with the agonists. In order to evaluate potential heterodimerization of the receptors, sensitized emission FRET experiments were performed in HEK293T cells using H1R-CFP and H2R-YFP. To our knowledge these findings may represent the first demonstration of agonist-induced heterodimerization of the H1R and H2R. In addition, we also show that the inhibition of the internalization process did not prevent receptor cross-desensitization which was mediated by GRK2. Our study provides new insights into the complex signaling network mediated by histamine and further knowledge for the rational use of its ligands.Fil: Alonso, Maria Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Fernández, Natalia Brenda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina;Fil: Notcovich, Cintia Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Monczor, Federico. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Simaan, May. National Institutes of Health; Estados Unidos de América;Fil: Baldi, Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Gutkind, J. Silvio. National Institutes of Health; Estados Unidos de América;Fil: Davio, Carlos Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Shayo, Carina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina

The head and neck cancer cell oncogenome: a platform for the development of precision molecular therapies

The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF-β in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration.Centro de Investigaciones Inmunológicas Básicas y Aplicada