Recent technological advances have revealed thousands of functional open reading frames (ORF) that have eluded reference genome annotations. These overlooked ORFs are found throughout the genome, in a Show more
Recent technological advances have revealed thousands of functional open reading frames (ORF) that have eluded reference genome annotations. These overlooked ORFs are found throughout the genome, in any reading frame of transcripts, mature or non-coding, and can overlap annotated ORFs in a different reading frame. The exploration of these novel ORFs in genomic datasets and of their role in genetic traits is hindered by a lack of software. Here, we present OpenVar, a genomic variant annotator that mends that gap and fosters meaningful discoveries. To illustrate the potential of OpenVar, we analysed all variants within SynMicDB, a database of cancer-associated synonymous mutations. By including non-canonical ORFs in the analysis, OpenVar yields a 33.6-fold, 13.8-fold and 8.3-fold increase in high impact variants over Annovar, SnpEff and VEP respectively. We highlighted an overlapping non-canonical ORF in the HEY2 gene where variants significantly clustered. OpenVar integrates non-canonical ORFs in the analysis of genomic variants, unveiling new research avenues to better understand the genotype-phenotype relationships. Show less
Beyond a well-recognized effect of KRAS mutations in determining de novo inefficacy of cetuximab (CTX) in metastatic colorectal cancer, we urgently need a biomarker signature for predicting CTX effica Show more
Beyond a well-recognized effect of KRAS mutations in determining de novo inefficacy of cetuximab (CTX) in metastatic colorectal cancer, we urgently need a biomarker signature for predicting CTX efficacy in KRAS wild-type (WT) tumors. CTX-adapted EGFR gene-amplified KRAS WT tumor cell populations were induced by stepwise-chronic exposure of A431 epidermoid cancer cells to CTX. Genome-wide analyses of 44K Agilent's whole human arrays were bioinformatically evaluated by Gene Set Enrichment Analysis (GSEA)-based screening of the KEGG pathway database. Molecular functioning of CTX was found to depend on: (i) The occurrence of a positive feedback loop on Epidermal Growth Factor Receptor (EGFR) activation driven by genes coding for EGFR ligands (e.g., amphiregulin); (ii) the lack of a negative feedback on mitogen-activated protein kinase (MAPK) activation regulated by dual-specificity phosphatases (e.g., DUSP6) and; (iii) the transcriptional status of gene pathways controlling the epithelial-to-mesenchymal transition (EMT) and its reversal (MET) program (actin cytoskeleton and cell-cell communication-e.g., keratins-focal adhesion signaling-e.g., integrins-and EMT-inducing cytokines - e.g., transforming growth factor-β). Quantitative real-time PCR, high-content immunostaining, and flow-cytometry analyses confirmed that CTX efficacy depends on its ability to promote: (i) Stronger cell-cell contacts by up-regulating the expression of the epithelial markers E-cadherin and occludin; (ii) down-regulation of the epithelial transcriptional repressors Zeb, Snail, and Slug accompanied by restoration of cortical F-actin; and (iii) complete prevention of the CD44(pos)/CD24(neg/low) mesenchymal immunophenotype. The impact of EGFR ligands/MAPK phosphatases gene transcripts in predicting CTX efficacy in KRAS WT tumors may be tightly linked with the ability of CTX to concurrently reverse the EMT status, a pivotal property of migrating cancer stem cells. Show less
Mitogen-activated protein kinase (MAPK) modules, composed of three protein kinases activated by successive phosphorylation, are involved in the signal transduction of a wide range of extracellular age Show more
Mitogen-activated protein kinase (MAPK) modules, composed of three protein kinases activated by successive phosphorylation, are involved in the signal transduction of a wide range of extracellular agents. In mammalian cells, mitogenic stimulation triggers the translocation of p42/p44MAPK from the cytoplasm to the nucleus, whereas the other protein kinases of the module remain cytosolic. Since MAPK has been shown to phosphorylate and activate nuclear targets, such as the transcription factor Elk1, it has been proposed, but not yet demonstrated, that MAPK nuclear translocation could represent a critical step in signal transduction. In this study, we sequestered p42/p44MAPK in the cytoplasm by the expression of a catalytically inactive form of cytoplasmic MAP kinase phosphatase (MKP-3/Pyst-1). Sequestering MAPK in the cytoplasm did not alter its activation or its ability to phosphorylate cytoplasmic substrates of MAPK (p90RSK1 or an engineered cytoplasmic form of Elk1). In contrast, prevention of MAPK nuclear translocation strongly inhibited Elk1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response to growth factors. Thus the relocalization of MAPK to the nucleus appears to be an important regulatory step for mitogen-induced gene expression and cell cycle re-entry. Show less