Canonical WNT signaling pathway and human AREG

AREG (Amphiregulin), BTC (beta-cellulin), EGF, EPGN (Epigen), EREG (Epiregulin), HBEGF, NRG1, NRG2, NRG3, NRG4 and TGFA (TGFalpha) constitute EGF family ligands for ERBB family receptors. Cetuximab (Erbitux), Pertuzumab (Omnitarg) and Trastuzumab (Herceptin) are anti-cancer drugs targeted to EGF family ligands, while Gefitinib (Iressa), Erlotinib (Tarceva) and Lapatinib (GW572016) are anti-cancer drugs targeted to ERBB family receptors. AREG and TGFA are biomarkers for Gefitinib non-responders. The TCF/LEF binding sites within the promoter region of human EGF family members were searched for by using bioinformatics and human intelligence (Humint). Because three TCF/LEF-binding sites were identified within the 5'-promoter region of human AREG gene, comparative genomics analyses on AREG orthologs were further performed. The EPGN-EREG-AREG-BTC cluster at human chromosome 4q13.3 was linked to the PPBP-CXCL segmental duplicons. AREG was the paralog of HBEGF at human chromosome 5q31.2. Chimpanzee AREG gene, consisting of six exons, was located within NW_105918.1 genome sequence. Chimpanzee AREG was a type I transmembrane protein showing 98.0% and 71.4% total amino-acid identity with human AREG and mouse Areg, respectively. Three TCF/LEF-binding sites within human AREG promoter were conserved in chimpanzee AREG promoter, but not in rodent Areg promoters. Primate AREG promoters were significantly divergent from rodent Areg promoters. AREG mRNA was expressed in a variety of human tumors, such as colorectal cancer, liver cancer, gastric cancer, breast cancer, prostate cancer, esophageal cancer and myeloma. Because human AREG was characterized as potent target gene of WNT/beta-catenin signaling pathway, WNT signaling activation could lead to Gefitinib resistance through AREG upregulation. AREG is a target of systems medicine in the field of oncology.     

Comparative integromics on Angiopoietin family members

Angiopoietin-1 (ANGPT1), Angiopoietin-4 (ANGPT4), VEGF, FGF2, FGF4, HGF, Ephrin, IL8 and CXCL12 (SFD1) are pro-angiogenic factors (angiogenic activators), while Angiopoietin-2 (ANGPT2), Angiostatin, Endostatin, Tumstatin, Canstatin, THBS1, THBS2, TNFSF15 (VEGI) and Vasohibin (VASH1) are anti-angiogenic factors (angiogenic inhibitors). ANGPT1 and ANGPT2 are ligands for TIE family receptor tyrosine kinases, TIE1 and TIE2 (TEK). Angiopoietin family consists of ANGPT1, ANGPT2, ANGPT4, ANGPTL1 (ANGPT3), ANGPTL2, ANGPTL3 (ANGPT5), ANGPTL4, ANGPTL5, ANGPTL6 and ANGPTL7. TCF/LEF binding sites within the promoter region of human Angiopoietin family members were searched for by using bioinformatics and human intelligence (Humint). Because four TCF/LEF-binding sites were identified within the human ANGPTL7 promoter, comparative genomics analyses on ANGPTL7 orthologs were further performed. ANGPTL7 gene at human chromosome 1p36.22 was located within intron 28 of FRAP1 gene encoding mTOR protein. Chimpanzee ANGPTL7 gene, consisting of five exons, was located within NW_101546.1 genome sequence. Chimpanzee ANGPTL7 showed 99.4% and 86.1% total-amino-acid identity with human ANGPTL7 and mouse Angptl7, respectively. Human ANGPTL7 mRNA was expressed in neural tissues, keratoconus cornea, trabecular meshwork, melanotic melanoma and uterus endometrial cancer, while mouse Angptl7 mRNA was expressed in four-cell embryo, synovial fibroblasts, thymus, uterus and testis. Four TCF/LEF-binding sites within human ANGPTL7 promoter were conserved in chimpanzee ANGPTL7 promoter; however, only an unrelated TCF/LEF-binding site occurred in mouse and rat Angptl7 promoters. Human ANGPTL7, characterized as potent target gene of WNT/ beta-catenin signaling pathway, is a pharmacogenomics target in the fields of oncology and regenerative medicine.     

Comparative genomics on FGF16 orthologs

We have previously reported comparative genomics analyses on FGF3, FGF4, FGF6, FGF7, FGF8, FGF10, FGF11, FGF17, FGF18, FGF19, FGF20, FGF22 and FGF23 genes. Here, we performed comparative genomics analyses on FGF1, FGF2, FGF5, FGF9, FGF12, FGF13, FGF14, FGF16 and FGF21 genes, and further characterized the FGF16 gene. Chimpanzee FGF16, chicken fgf16, and zebrafish fgf16 genes were identified within NW_121938.1, NW_060344.1, and CR855117.3 genome sequences, respectively. Chimpanzee FGF16 (207 aa), chicken fgf16 (207 aa), and zebrafish fgf16 (203 aa) showed 100%, 89.9%, and 79.2% total amino-acid identity with human FGF16. Because FGF16, FGF9, and FGF20 constitute FGF subfamily without N-terminal signal peptide, we next searched for uncharacterized FGF9 or FGF20 orthologs. Zebrafish fgf9 gene was identified within BX927112.11 genome sequence, and chicken fgf20 gene within NW_060349.1 genome sequence. Although N-terminal part was divergent, middle and C-terminal parts were well conserved among vertebrate FGF16, FGF9 and FGF20 orthologs. Phylogenetic analyses revealed that zebrafish fgf9 and fgf20 were more related to each other than to their chicken or mammalian orthologs. TCF/LEF binding site and TATA box were well conserved among the human FGF16, rat Fgf16, and mouse Fgf16 promoters. Because nuclear complex consisting of TCF/LEF (TCF1, TCF3, TCF4 or LEF1), beta-catenin, PYGO (PYGO1 or PYGO2) and Legless (BCL9 or BCL9L) binds to the TCF/LEF-binding site to up-regulate WNT/beta-catenin target genes, FGF16 gene was characterized as the evolutionarily conserved target of the WNT/beta-catenin signaling pathway, just like FGF18 and FGF20 genes. These facts indicate that FGF16, FGF18 and FGF20 are pharmacogenomics targets in the field of oncology and regenerative medicine.     

JAG1-Mediated Notch Signaling Regulates Secretory Cell Differentiation of the Human Airway Epithelium

Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.     

Anti-Jagged-1 immunotherapy in cancer

Notch signaling is a highly conserved pathway and it plays an essential role in regulating cellular proliferation, differentiation, and apoptosis. The human Notch family includes four receptors, Notch 1–4, and five ligands, delta-like ligand 1 (DLL1), delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), Jagged-1 (JAG1), and Jagged-2 (JAG2). It is widely known, that Notch signaling components are often mutated and have deregulated expression in many types of cancer and other diseases. Thus, various therapeutic approaches targeting receptors and ligands of the Notch pathway are being investigated. Human JAG1 is closely related to tumor biology among the Notch ligands, and recent studies have shown potential for monoclonal antibodies targeting JAG1 in cancer therapy. Therefore, this review focuses on current reports on the significance of JAG1 directed cancer treatment, emphasizing immunotherapy.     

Notch signalling suppresses regulatory T‐cell function in murine experimental autoimmune uveitis

Autoimmune uveitis is an intraocular inflammatory disorder in developed countries. Understanding the mechanisms underlying the development and modulation of immune reaction in uveitic eyes is critical for designing therapeutic interventions. Here we investigated the role of Notch signalling in regulatory T‐cell (Treg cell) function during experimental autoimmune uveitis (EAU). Using the Foxp3‐GFP reporter mouse strain, the significance of Notch signalling for the function of infiltrating Treg cells was characterized in an EAU model. We found that infiltrating Treg cells substantially expressed Notch‐1, Notch‐2, JAG1 and DLL1 in uveitic eyes. Activation of Notch signalling, represented by expression of HES1 and HES5, was enhanced in infiltrating Treg cells. Treatment with JAG1 and DLL1 down‐regulated Foxp3 expression and immunosuppressive activity of isolated infiltrating Treg cells in vitro, whereas neutralizing antibodies against JAG1 and DLL1 diminished Notch ligand‐mediated negative effects on Treg cells. To investigate the significance of Notch signalling for Treg cell function in vivo, lentivirus‐derived Notch short hairpin RNAs were transduced into in vitro expanded Treg cells before adoptive transfer of Treg cells into EAU mice. Transfer of Notch‐1‐deficient Treg cells remarkably reduced pro‐inflammatory cytokine production and inflammatory cell infiltration in uveitic eyes. Taken together, Notch signalling negatively modulates the immunosuppressive function of infiltrating Treg cells in mouse EAU.     

Comparative genomics on FGF8, FGF17, and FGF18 orthologs

FGF and WNT signaling pathways network together during embryogenesis and carcinogenesis. Among 22 FGF family members within human and rodents genomes, FGF20 orthologs are evolutionarily conserved targets of the WNT/beta-catenin signaling pathway. FGF8, FGF17, and FGF18 constitute one of FGF subfamilies. Here, comparative proteomics and comparative genomics analyses on FGF8, FGF17, and FGF18 orthologs were performed. Rat Fgf8 and Fgf17 genes, consisting of five exons, were located within AC096326.7 and AC097410.12 genome sequences, respectively. FGF8, FGF17, and FGF18 orthologs were FGF family members with the N-terminal signal peptide. Human FGF8 isoform F showed 90.6% total-amino-acid identity with rat Fgf8 (268 aa). Human FGF17 showed 98.6% total-amino-acid identity with rat Fgf17 (216 aa). Human FGF18 also showed 98.6 total-amino-acid identity with rat Fgf18. FBXW1 (betaTRCP1 or BTRC1)-FGF8-NPM3 locus at human chromosome 10q24.32, FBXW11 (betaTRCP2 or BTRC2)-FGF18-NPM1 locus at human chromosome 5q35.1, and FGF17-NPM2 locus at human chromosome 8p21.3 were paralogous regions within the human genome. FGF8 mRNA was expressed in DMSO-treated embryonic stem (ES) cells. FGF17 mRNA was expressed in ES cells differentiated to an early endodermal phenotype. FGF18 mRNA was expressed in fetal lung, fetal heart, lung carcinoid, colorectal cancer, and ovarian cancer. FGF18 promoter with double TCF/LEF binding sites rather than FGF8 promoter and FGF17 promoter was more conserved between human and rodents. These facts indicate that FGF18 orthologs were evolutionarily conserved targets of the WNT/beta-catenin signaling pathway.