[Relapsed/refractory Philadelphia chromosome-positive acute lymphoblastic leukemia responding to retreatment with inotuzumab ozogamicin].

A 72-year-old man with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) was treated with dasatinib (week1: 50 mg/day, week2: 70 mg/day, week3-: 100 mg/day) and prednisolone from June 2017. However, in January 2018, it relapsed with the T315I mutation. Although the treatment was changed to ponatinib 30 mg/day, he experienced a second relapse in June 2018. Following confirmation of CD22 positivity, he was treated with three cycles of inotuzumab ozogamicin (InO), resulting in CR. He was CR for 2.9 years before relapsing for the third time in May 2021. Because the patient was still CD22-positive, InO was given again, and the patient achieved CR at the end of the second cycle. We had a case where re-administering InO was effective as a salvage therapy for relapsed/refractory Ph+ALL (r/r Ph+ALL) in an elderly patient.

Noise stability of synchronization and optimal network structures.

We provide a theoretical framework for quantifying the expected level of synchronization in a network of noisy oscillators. Through linearization around the synchronized state, we derive the following quantities as functions of the eigenvalues and eigenfunctions of the network Laplacian using a standard technique for dealing with multivariate Ornstein-Uhlenbeck processes: the magnitude of the fluctuations around a synchronized state and the disturbance coefficients αi that represent how strongly node i disturbs the synchronization. With this approach, we can quantify the effect of individual nodes and links on synchronization. Our theory can thus be utilized to find the optimal network structure for accomplishing the best synchronization. Furthermore, when the noise levels of the oscillators are heterogeneous, we can also find optimal oscillator configurations, i.e., where to place oscillators in a given network depending on their noise levels. We apply our theory to several example networks to elucidate optimal network structures and oscillator configurations.

Integrative genomic analyses on GLI1: positive regulation of GLI1 by Hedgehog-GLI, TGFbeta-Smads, and RTK-PI3K-AKT signals, and negative regulation of GLI1 by Notch-CSL-HES/HEY, and GPCR-Gs-PKA signals.

GLI family members are zinc-finger transcription factors, which are involved in embryogenesis and carcinogenesis through transcription regulation of GLI1, CCND1, CCND2, FOXA2, FOXC2, RUNX2, SFRP1, and JAG2. GLI1 transcription is upregulated in a variety of human tumors, such as basal cell carcinoma, lung cancer, breast cancer, gastric cancer, pancreatic cancer, and esophageal cancer. Hedgehog signaling via Smoothened cascade and receptor tyrosine kinase (RTK) signaling via PI3K-AKT cascade induce stabilization of GLI1 protein, whereas G-protein coupled receptor (GPCR) signaling via Gs-PKA cascade induces degradation of GLI1 protein. Here we report integrative genomic analyses of the GLI1 gene. The GLI1 and ARHGAP9 genes are located in a tail-to-tail manner with overlapping 3'-ends. ARHGAP9 was expressed in bone marrow, spleen, thymus, monocytes, and macrophages, whereas GLI1 was almost undetectable in normal tissues or cells with predominant ARHGAP9 expression. Because overlapping sense and anti-sense transcripts are annealed to each other to give rise to double-stranded RNAs functioning as endogenous RNAi, GLI1 expression might be negatively regulated by ARHGAP9 transcripts. GLI-binding element with one base substitution at the +1589-bp position from the transcriptional start site (TSS) of the human GLI1 gene was completely conserved in chimpanzee GLI1, mouse Gli1, and rat Gli1 genes. Ten Smad-binding elements, double E-boxes for EMT regulators, and double N-boxes for HES/HEY family members within intron 1 of the human GLI1 gene were also conserved in mammalian GLI1 orthologs. GLI1 transcription is upregulated due to Hedgehog, and TGFbeta signaling activation, whereas GLI1 transcription is downregulated due to Snail/Slug, and Notch signaling activation. Together these facts indicate that Hedgehog, TGFbeta, and RTK signals positively regulate GLI1, and that Notch, and GsPCR signals negatively regulate the GLI1.

FGFR2-related pathogenesis and FGFR2-targeted therapeutics (Review).

FGFR2 gene at human chromosome 10q26 encodes FGFR2b and FGFR2c isoforms functioning as FGF receptors with distinct expression domain and ligand specificity. FGFR2 plays oncogenic and anti-oncogenic roles in a context-dependent manner. Single nucleotide polymorphisms (SNPs) within intron 2 of FGFR2 gene are associated with breast cancer through allelic FGFR2 upregulation. Missense mutations or copy number gains of FGFR2 gene occur in breast cancer and gastric cancer to activate FGFR2 signaling. Aberrant FGFR2 signaling activation induces proliferation and survival of tumor cells. The class switch from FGFR2b to FGFR2c occurs during progression of prostate cancer and bladder cancer because of spliceosome dysregulation. In addition, epidermal Fgfr2b knockout mice show increased sensitivity to chemical carcinogenesis partly due to the failure of Nfe2l2 (Nrf2)-mediated detoxification of reactive oxygen species (ROS). Loss of FGFR2b signaling induces epithelial-to-mesenchymal transition (EMT) and unruly ROS. FGFR2 signaling dysregulation due to the accumulation of epigenetic modifications and genetic alterations during chronic inflammation, smoking, increased caloric uptake, and decreased exercise leads to carcinogenesis. PD173074, SU5402, AZD2171, and Ki23057 are small-molecule FGFR inhibitors. Human antibody, peptide mimetic, RNA aptamer, siRNA, and synthetic microRNA (miRNA) are emerging technologies to be applied for cancer therapeutics targeted to FGFR2. Because novel sequence technology and peta-scale super-computer are opening up the sequence era following the genome era, personalized medicine prescribing targeted drugs based on germline and/or somatic genomic information is coming reality. Application of FGFR2 inhibitors for cancer treatment in patients with FGFR2 mutation or gene amplification is beneficial; however, that for cancer prevention in people with FGFR2 risk allele might be disadvantageous due to the impediment of a cytoprotective mechanism against oxidative stress.

Integrative genomic analyses on GLI2: mechanism of Hedgehog priming through basal GLI2 expression, and interaction map of stem cell signaling network with P53.

Hedgehog-binding to Patched family receptors results in Smoothened-mediated activation of MAP3K10 (MST) and inactivation of SUFU. MAP3K10-induced DYRK2 phosphorylation combined with SUFU inhibition results in the stabilization and nuclear accumulation of GLI2 for transcriptional activation of GLI1, CCND1, CCND2, FOXA2, FOXC2, FOXP3, FOXQ1, RUNX2, and JAG2. Here, integrative genomic analyses on GLI2 orthologs were carried out. Rat Gli2 complete coding sequence was determined by assembling nucleotide sequences of exons 1, 2, and 5'-truncated rat Gli2 RefSeq (NM_001107169.1). GLI2 orthologs were more related to GLI3 orthologs than to GLI1 orthologs lacking the N-terminal repressor domain. betaTRCP1 (FBXW1)-binding DSYxxxS motif was conserved in GLI2 and GLI3 orthologs, while betaTRCP2 (FBXW11)-binding DSGxxxxxxxxxS motif in GLI2 and GLI1 orthologs. Human GLI2 mRNA was expressed in ES cells, NT2 cells, fetal lung, fetal heart, regenerating liver, gastric cancer, and other tumors. Mouse Gli2 mRNA was expressed in unfertilized egg, ES cells, and EG cells. Tandem RRRCWWGYYY motifs for P53, P63 or P73, and also four conserved bHLH-binding sites were identified within GLI2 proximal promoter region. Interaction map of P53 and stem cell signaling network were then constructed. P53-induced NOTCH1 upregulation leads to HES1, HES5, HEY1, HEY2 or HEYL upregulation for the repression of tissue specific bHLH transcriptional activators. DYRK2 functions as a positive regulator of P53-mediated apoptosis, and also as a negative regulator of the Hedgehog signaling cascade. GLI2 expression is regulated based on the balance of P53, Notch, and TGF-beta signaling, and Hedgehog signaling activation results in cell survival and proliferation due to transcriptional activation of Hedgehog-target genes, and also partly due to perturbation of P53-mediated transcriptional regulation.

Hedgehog signaling, epithelial-to-mesenchymal transition and miRNA (review).

SHH, IHH, and DHH are lipid-modified secreted proteins binding to Patched receptors, and CDON, BOC or GAS1 co-receptors. In the absence of Hedgehog signaling, GLI1 is transcriptionally repressed, GLI2 is phosphorylated by GSK3 and CK1 for the FBXW11 (betaTRCP2)-mediated degradation, and GLI3 is processed to a cleaved repressor. In the presence of Hedgehog signaling, Smoothened is relieved from Patched-mediated suppression due to the Hedgehog-dependent internalization of Patched, which leads to MAP3K10 (MST) activation and SUFU inactivation for the stabilization and nuclear accumulation of GLI family members. GLI activators then upregulate CCND1, CCND2 for cell cycle acceleration, FOXA2, FOXC2, FOXE1, FOXF1, FOXL1, FOXP3, POU3F1, RUNX2, SOX13, TBX2 for cell fate determination, JAG2, INHBC, and INHBE for stem cell signaling regulation. Hedgehog signals also upregulate SFRP1 in mesenchymal cells for WNT signaling regulation. Epithelial-to-mesenchymal transition (EMT) during embryogenesis, adult tissue homeostasis and carcinogenesis is characterized by class switch from E-cadherin to N-cadherin. SNAI1 (Snail), SNAI2 (Slug), SNAI3, ZEB1, ZEB2 (SIP1), KLF8, TWIST1, and TWIST2 are EMT regulators repressing CDH1 gene encoding E-cadherin. Hedgehog signals induce JAG2 upregulation for Notch-CSL-mediated SNAI1 upregulation, and also induce TGFbeta1 secretion for ZEB1 and ZEB2 upregulation via TGFbeta receptor and NF-kappaB. TGFbeta-mediated downregulation of miR-141, miR-200a, miR-200b, miR-200c, miR-205, and miR-429 results in upregulation of ZEB1 and ZEB2 proteins. Hedgehog signaling activation indirectly leads to EMT through FGF, Notch, TGFbeta signaling cascades, and miRNA regulatory networks. miRNAs targeted to stem cell signaling components or EMT regulators are potent drug targets; however, off-target effects should be strictly controlled before clinical application of synthetic miRNA. Peptide mimetic and RNA aptamer could also be utilized as Hedgehog signaling inhibitors or EMT suppressors.

Comparative integromics on JMJD2A, JMJD2B and JMJD2C: preferential expression of JMJD2C in undifferentiated ES cells.

Fertilized egg or totipotent zygote undergoes cleavage divisions to form a blastocyst, consisting of outer trophoectoderm cells and inner cell mass with pluripotent primitive ectoderm cells. Epigenetic reprogramming, erasure and maintenance of epigenetic modification, occurs during early embryogenesis. In 2004, we identified and characterized JMJD2A/JHDM3A, JMJD2B, JMJD2C, JMJD2D, JMJD2E and JMJD2F. JMJD2A, JMJD2B and JMJD2C share the common domain architecture with JmjN, JmjC, two PHD, and two TUDOR domains. In 2006, other groups characterized JMJD2 family members as the H3K9 and/or H3K36 histone demethylases. Here, comparative integromics analyses on JMJD2A, JMJD2B and JMJD2C were carried out. Mouse Jmjd2a was expressed in fertilized egg and 2-cell embryos, while human JMJD2A was expressed in undifferentiated and differentiated ES cells. AP1-binding site and six bHLH-binding sites within intron 13 of human JMJD2A gene were conserved in mouse Jmjd2a gene. Mouse Jmjd2b was expressed in 8-cell embryos and undifferentiated ES cells, while human JMJD2B was expressed in undifferentiated and differentiated ES cells. Two GATA-binding sites within intron 6 of human JMJD2B gene were conserved in mouse Jmjd2b gene. Mouse Jmjd2c and human JMJD2C were preferentially expressed in undifferentiated ES cells. Four NANOG-binding sites, one TCF/ LEF-binding site, and one bHLH-binding site were located within evolutionary conserved region at the 3'-flanking region of human JMJD2C gene. NANOG- TCF/LEF-, and bHLH-binding sites within the 3'-flanking region of human JMJD2C gene were conserved in chimpanzee, cow, mouse and rat JMJD2C othologs. Together these facts indicate that JMJD2C is the evolutionarily conserved target of Homeo-domain transcription factor NANOG, and that JMJD2C is the histone demethylase implicated in the epigenetic reprogramming during the early embryogenesis.

Comparative genomics on PROM1 gene encoding stem cell marker CD133.

Stem cells are characterized by self-renewal and multipotency to produce multiple lineages of progenitor and differentiated cells. PROM1 gene encodes CD133 protein, which is a cell surface marker of hematopoietic stem cells, prostatic epithelial stem cells, pancreatic stem cells, leukemic stem cells, liver cancer stem cells, and colorectal cancer stem cells. Here, comparative integromics analyses on PROM1 orthologs were performed. Human PROM1 RefSeq NM_006017.1 was a truncated transcript, while AK027422.1 was the representative human PROM1 cDNA. Chimpanzee PROM1 gene, consisting of 27 exons, was identified within NW_001234057.1 genome sequence. Chimpanzee 5-transmembrane protein CD133 showed 99.2% and 60.9% total-amino-acid identity with human and mouse CD133 orthologs, respectively. Only 2 of 8 Asn-linked glycosylation sites in primate CD133 orthologs were conserved in rodent CD133 orthologs. Comparative proteomics revealed that CD133 orthologs were relatively divergent between primates and rodents. PROM1 mRNA was expressed in human embryonic stem (ES) cells, trachea, small intestine, NT2 cells, diffuse-type gastric cancer, and colorectal cancer. Human PROM1 mRNA transcribed from exon 1A was the major transcript. Comparative genomics revealed that the region around exon 1A corresponding to 5'-UTR of human PROM1 mRNA was not conserved in mouse and rat. Intron 2 of PROM1 orthologs was relatively well conserved among mammals. Tandem TCF/LEF-binding sites with 7-bp spacing within intron 2 were conserved among human, chimpanzee, mouse, and rat PROM1 orthologs. Together these facts indicate that canonical WNT signaling activation is implicated in CD133 expression in ES cells, adult stem cells, and cancer stem cells.

Conserved POU-binding site linked to SP1-binding site within FZD5 promoter: Transcriptional mechanisms of FZD5 in undifferentiated human ES cells, fetal liver/spleen, adult colon, pancreatic islet, and diffuse-type gastric cancer.

Canonical WNT signals are transduced through Frizzled (FZD) family receptor and LRP5/LRP6 co-receptor to upregulate FGF20, JAG1, DKK1, WISP1, CCND1 and MYC genes for cell-fate determination, while non-canonical WNT signals are transduced through FZD family receptor and ROR2/PTK7/RYK co-receptor to activate RHOA/RHOU/RAC/CDC42, JNK, PKC, NLK and NFAT signaling cascades for the regulation of tissue polarity, cell movement, and adhesion. We previously reported molecular cloning and characterization of human FZD5, which showed six amino-acid substitutions with human Hfz5. FZD5, functioning as WNT5A receptor, is the key molecule in the fields of oncology, regenerative medicine, cardiology, rheumatology, diabetology, and gastroenterology. Here, comparative integromics analyses on FZD5 orthologs were performed by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee FZD5 and cow Fzd5 genes were identified within NW_104292.1 and AC166656.2 genome sequences, respectively. FZD5 orthologs were seven-transmembrane proteins with extracellular Frizzled domain, leucine zipper motif around the 5th transmembrane domain, and cytoplasmic DVL- and PDZ-binding motifs. Ser523 and Ser529 around the DVL-binding motif of FZD5 orthologs were putative aPKC phosphorylation sites. POU5F1 (OCT4)-binding site linked to SP1-binding site within the 5'-promoter region of human FZD5 gene was evolutionarily conserved among mammalian FZD5 orthologs. POU5F1 was more related to POU2F and POU3F subfamily members. POU5F1 was preferentially expressed in undifferentiated human embryonic stem (ES) cells, pancreatic islet, and diffuse-type gastric cancer. POU2F1 (OCT1) was expressed in ES cells, fetal liver/spleen, adult colon, POU2F2 in ES cells, fetal liver/spleen, and POU2F3 in diffuse-type gastric cancer. Multiple SP1/KLF family members, other than KLF2 or KLF4, were expressed in undifferentiated human ES cells. Together, these facts indicate that POU5F1 and POU2F subfamily members play a pivotal role for the FZD5 expression in undifferentiated human ES cells, fetal liver/spleen, adult colon, pancreatic islet, and diffuse-type gastric cancer.

Hedgehog signaling pathway and gastrointestinal stem cell signaling network (review).

Hedgehog, BMP/TGFbeta, FGF, WNT and Notch signaling pathways constitute the stem cell signaling network, which plays a key role in a variety of processes, such as embryogenesis, maintenance of adult tissue homeostasis, tissue repair during chronic persistent inflammation, and carcinogenesis. Sonic hedgehog (SHH), Indian hedgehog (IHH) and Desert hedgehog (DHH) bind to PTCH1/PTCH or PTCH2 receptor to release Smoothened (SMO) signal transducer from Patched-dependent suppression. SMO then activates STK36 serine/threonine kinase to stabilize GLI family members and to phosphorylate SUFU for nuclear accumulation of GLI. Hedgehog signaling activation leads to GLI-dependent transcriptional activation of target genes, such as GLI1, PTCH1, CCND2, FOXL1, JAG2 and SFRP1. GLI1-dependent positive feedback loop combined with PTCH1-dependent negative feedback loop gives rise to transient proliferation of Hedgehog target cells. Iguana homologs (DZIP1 and DZIP1L) and Costal-2 homologs (KIF7 and KIF27) are identified by comparative integromics. SHH-dependent parietal cell proliferation is implicated in gastric mucosal repair during chronic Helicobacter pylori infection. BMP-RUNX3 signaling induces IHH expression in surface differentiated epithelial cells of stomach and intestine. Hedgehog signals from epithelial cells then induces FOXL1-mediated BMP4 upregulation in mesenchymal cells. Hedgehog signaling is frequently activated in esophageal cancer, gastric cancer and pancreatic cancer due to transcriptional upregulation of Hedgehog ligands and epigenetic silencing of HHIP1/HHIP gene, encoding the Hedgehog inhibitor. However, Hedgehog signaling is rarely activated in colorectal cancer due to negative regulation by the canonical WNT signaling pathway. Hedgehog signaling molecules or targets, such as SHH, IHH, HHIP1, PTCH1 and GLI1, are applied as biomarkers for cancer diagnostics, prognostics and therapeutics. Small-molecule inhibitors for SMO or STK36 are suitable to be used for treatment of Hedgehog-dependent cancer.

Comparative integromics on FAT1, FAT2, FAT3 and FAT4.

WNT5A, WNT5B, WNT11, FZD3, FZD6, VANGL1, VANGL2, DVL1, DVL2, DVL3, PRICKLE1, PRICKLE2, ANKRD6, NKD1, NKD2, DAAM1, DAAM2, CELSR1, CELSR2, CELSR3, ROR1 and ROR2 are planar cell polarity (PCP) signaling molecules implicated in the regulation of cellular polarity, convergent extension, and invasion. FAT1, FAT2, FAT3 and FAT4 are Cadherin superfamily members homologous to Drosophila Fat, functioning as a positive regulator of PCP in the Drosophila wing. Complete coding sequence (CDS) for human FAT1 (NM_005245.3) and FAT2 (NM_001447.1) are available, while artificial CDS for human FAT3 (XM_926199 and XM_936538) and partial CDS for FAT4 (NM_024582.2). Here, complete CDS of human FAT3 and FAT4 were determined by using bioinformatics and human intelligence (Humint). FAT3 gene, consisting of 26 exons, encoded a 4557-aa protein with extracellular 33 Cadherin repeats, one Laminin G (LamG) domain and two EGF domains. FAT4 gene encoded a 4924-aa protein with extracellular 34 Cadherin repeats, two LamG domains and three EGF domains. Cytoplasmic VCSVxPxLP and SDYxS motifs were identified as novel motifs conserved among FAT1, FAT2 and FAT3 orthologs. Domain architecture comparison and phylogenetic analysis revealed that FAT1, FAT2 and FAR3 were divergent from FAT4. FAT1-MTNR1A locus at 4q35.2 and FAT3-MTNR1B locus at 11q14.3-q21 were paralogous regions within the human genome. FAT1 mRNA was expressed in embryonic stem (ES) cells, neural tissues, gastric cancer, pancreatic cancer, colorectal cancer, breast cancer, lung cancer and brain tumors. FAT2 mRNA was expressed in infant brain, cerebellum, gastric cancer, pancreatic cancer, ovarian cancer, esophageal cancer, skin squamous cell carcinoma, head and neck cancer. FAT3 mRNA was expressed in ES cells, primitive neuroectoderm, fetal brain, infant brain, adult neural tissues and prostate. FAT4 mRNA was expressed in fetal brain, infant brain, brain tumor and colorectal cancer. FAT family members were revealed to be targets of systems medicine in the fields of oncology and neurology.

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.

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 VEGF family members.

VEGF, Hedgehog, FGF, Notch, and WNT signaling pathways network together for vascular remodeling during embryogenesis, tissue regeneration, and carcinogenesis. VEGFA (VEGF), VEGFB, VEGFC, VEGFD (FIGF) and PGF (PlGF) are VEGF family ligands for receptor tyrosine kinases, including VEGFR1 (FLT1), VEGFR2 (KDR) and VEGFR3 (FLT4). Bevacizumab (Avastin), Sunitinib (Sutent) and Sorafenib (Nexavar) are anti-cancer drugs targeted to VEGF signaling pathway. TCF/LEF binding sites within the promoter region of human VEGF family members were searched for by using bioinformatics and human intelligence (Humint). Because four TCF/LEF-binding sites were identified within the 5'-promoter region of human VEGFD gene within AC095351.5 genome sequence, comparative genomics analyses on VEGFD orthologs were further performed. ASB9-ASB11-VEGFD locus at human chromosome Xp22.2 and ASB5-VEGFC locus at human chromosome 4q34 were paralogous regions within the human genome. Human VEGFD mRNA was expressed in lung, small intestine, uterus, breast, neural tissues, and neuroblastoma. Mouse Vegfd mRNA was expressed in kidney, pregnant oviduct, and neural tissues. Chimpanzee VEGFD promoter, cow Vegfd promoter, mouse Vegfd promoter and rat Vegfd promoter were identified within NW_121675.1, AC161065.2, AL732475.6 and AC130036.3 genome sequences, respectively. Three out of four TCF/LEF-binding sites within human VEGFD promoter were conserved in chimpanzee VEGFD promoter, and one in cow Vegfd promoter. TCF/LEF-binding site, not conserved in human VEGFD promoter, occurred in cow, mouse and rat Vegfd promoters. At least five out of six bHLH-binding sites within human VEGFD proximal promoter region were conserved in chimpanzee VEGFD proximal promoter region, while only one in cow Vegfd proximal promoter region. Together these facts indicate that relatively significant promoter evolution occurred among mammalian VEGFD orthologs. Human VEGFD was characterized as a potent target gene of WNT/beta-catenin signaling pathway. VEGFD, implicated in angiogenesis and lymphatic metastasis, is a pharmacogenomics target in the field of oncology.

Comparative integromics on Ephrin family.

EFNA1, EFNA2, EFNA3, EFNA4, EFNA5, EFNB1, EFNB2 and EFNB3 are EFN family ligands for EPH family receptors. EFN/EPH signaling pathway networks with the WNT signaling pathway during embryogenesis, tissue regeneration, and carcinogenesis. Comparative genomics analyses on EFNB1, EFNB2 and EFNB3 were performed by using bioinformatics and human intelligence (humint). EFNB1 mRNA was expressed in human embryonic stem (ES) cells, neural tissues, diffuse type gastric cancer, pancreatic cancer, colon cancer, brain tumors and esophageal cancer, EFNB2 mRNA in human ES cells, neural tissues and colon cancer, EFNB3 mRNA in human ES cells, neural tissues, brain tumors, pancreatic cancer and colon cancer. Because triple TCF/LEF-binding sites were identified within the 5'-promoter region of human EFNB3 gene, comparative genomics analyses on EFNB3 orthologs were further performed. Chimpanzee EFNB3 gene, consisting of five exons, was identified within AC164921.3 genome sequence. AY421228.1 was not a correct coding sequence for chimpanzee EFNB3. Chimpanzee EFNB3 gene was found to encode a 340-amino-acid protein showing 99.4% and 96.6% total-amino-acid identity with human EFNB3 and mouse Efnb3, respectively. Three TCF/LEF-binding sites within human EFNB3 promoter were conserved in chimpanzee EFNB3 promoter, and the second TCF/LEF-binding site in rodent Efnb3 promoters. CpG hypermethylation of EFNB3 promoter with 63.2% GC content as well as deletion of EFNB3 gene closely linked to TP53 tumor suppressor gene at human chromosome 17p13.1 should be investigated to elucidate the mechanism of infrequent EFNB3 upregulation in human colorectal cancer. EFNB3, identified as potential transcriptional target of WNT/beta-catenin signaling pathway, is a pharmacogenomics target in the fields of regenerative medicine and oncology.

Comparative integromics on BMP/GDF family.

WNT, Notch, FGF, Hedgehog and BMP signaling pathways network together during embryogenesis, tissue regeneration, and carcinogenesis. BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP10, BMP15, AMH, GDF1, GDF2, GDF3, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, and GDF15 are BMP/GDF family genes within the human genome; however, transcriptional regulation of BMP/GDF family members by the canonical WNT signaling pathway remains unclear. We searched for the TCF/LEF-binding site within the promoter region of BMP/GDF family genes by using bioinformatics and human intelligence. Because four TCF/LEF-binding sites were identified within human GDF10 promoter, comparative genomics analyses on GDF10 orthologs were further performed. Chimpanzee GDF10 gene, encoding a 477-amino-acid protein, was identified within NW_112875.1 genome sequence. AY412135.1 was not the correct coding sequence for chimpanzee GDF10. Chimpanzee GDF10 showed 99.2%, 83.2% and 47.4% total amino-acid identity with human GDF10, mouse Gdf10 and human BMP3, respectively. RASGEF1A-GDF10-PRKG1 locus at human chromosome 10q11 and BMP3-PRKG2-RASGEF1B locus at human chromosome 4q21 were paralogous regions with insertions/deletions and recombination. Human GDF10 mRNA was expressed in fetal cochlea, fetal lung, testis, retina, pineal gland, other neural tissues, head and neck tumors, while mouse Gdf10 mRNA was expressed in fetal liver, inner ear, cerebellum, other neural tissues, prostate and blood vessels. Four TCF/LEF-binding sites in human GDF10 promoter were conserved in chimpanzee GDF10 promoter, but not in the mouse Gdf10 promoter; however, another TCF/LEF-binding site occurred in mouse Gdf10 promoter. Four bHLH-binding sites in human GDF10 promoter were conserved in chimpanzee GDF10 promoter, but only one in mouse Gdf10 promoter. Primate GDF10 promoters were divergent from mouse Gdf10 promoter. Because GDF10 was characterized as a potential target of canonical WNT signaling pathway in neural tissues, GDF10 is one of the targets of systems medicine, especially in the field of regenerative medicine.

FGF signaling inhibitor, SPRY4, is evolutionarily conserved target of WNT signaling pathway in progenitor cells.

WNT, FGF and Hedgehog signaling pathways network together during embryogenesis, tissue regeneration, and carcinogenesis. FGF16, FGF18, and FGF20 genes are targets of WNT-mediated TCF/LEF-beta-catenin-BCL9/BCL9L-PYGO transcriptional complex. SPROUTY (SPRY) and SPRED family genes encode inhibitors for receptor tyrosine kinase signaling cascades, such as those of FGF receptor family members and EGF receptor family members. Here, transcriptional regulation of SPRY1, SPRY2, SPRY3, SPRY4, SPRED1, SPRED2, and SPRED3 genes by WNT/beta-catenin signaling cascade was investigated by using bioinformatics and human intelligence (humint). Because double TCF/LEF-binding sites were identified within the 5'-promoter region of human SPRY4 gene, comparative genomics analyses on SPRY4 orthologs were further performed. SPRY4-FGF1 locus at human chromosome 5q31.3 and FGF2-NUDT6-SPATA5-SPRY1 locus at human chromosome 4q27-q28.1 were paralogous regions within the human genome. Chimpanzee SPRY4 gene was identified within NW_107083.1 genome sequence. Human, chimpanzee, rat and mouse SPRY4 orthologs, consisting of three exons, were well conserved. SPRY4 gene was identified as the evolutionarily conserved target of WNT/beta-catenin signaling pathway based on the conservation of double TCF/LEF-binding sites within 5'-promoter region of mammalian SPRY4 orthologs. Human SPRY4 mRNA was expressed in embryonic stem (ES) cells, brain, pancreatic islet, colon cancer, head and neck tumor, melanoma, and pancreatic cancer. WNT signaling activation in progenitor cells leads to the growth regulation of progenitor cells themselves through SPRY4 induction, and also to the growth stimulation of proliferating cells through FGF secretion. Epigenetic silencing and loss-of-function mutations of SPRY4 gene in progenitor cells could lead to carcinogenesis. SPRY4 is the pharmacogenomics target in the fields of oncology and regenerative medicine.

Comparative genomics on HHIP family orthologs.

Hedgehog, FGF, VEGF, and Notch signaling pathways network together for vascular remodeling during embryogenesis and carcinogenesis. HHIP1 (HHIP) is an endogenous antagonist for SHH, IHH, and DHH. Here, comparative integromics analyses on HHIP family members were performed by using bioinformatics and human intelligence. HHIP1, HHIP2 (HHIPL1 or KIAA1822) and HHIP3 (HHIPL2 or KIAA1822L) constitute human HHIP gene family. Rat Hhip1, Hhip2, and Hhip3 genes were identified within AC107504.4, AC094820.6, and AC134264.2 genome sequences, respectively. HHIP-homologous (HIPH) domain with conserved 18 Cys residues was identified as the novel domain conserved among mammalian HHIP1, HHIP2, and HHIP3 orthologs. HHIP1 mRNA was expressed in coronary artery endothelial cells, prostate, and rhabdomyosarcoma. HHIP2 mRNA was expressed in trabecular bone cells. HHIP3 mRNA was expressed in testis, thyroid gland, osteoarthritic cartilarge, pancreatic cancer, and lung cancer. Promoters of HHIP family genes were not well conserved between human and rodents. Although GLI-, CSL-, and HES/HEY-binding sites were not identified, eleven bHLH-binding sites were identified within human HHIP1 promoter. Expression of HES/HEY family members, including HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2 and HEYL, in coronary artery endothelial cells was not detected in silico. Up-regulation of HHIP1 due to down-regulation of Notch-CSL-HES/HEY signaling cascade repressing bHLH transcription factors results in down-regulation of the Hedgehog-VEGF-Notch signaling cascade. On the other hand, down-regulation of HHIP1 due to up-regulation of Notch signaling in vascular endothelial cells during angiogenesis results in up-regulation of the Hedgehog-VEGF-Notch signaling cascade. Because HHIP1 is the key molecule for vascular remodeling, HHIP1 is the pharmacogenomics target in the fields of oncology and vascular medicine.

WNT antagonist, SFRP1, is Hedgehog signaling target.

Hedgehog and WNT signaling pathways network together during embryogenesis and carcinogenesis. Hedgehog signaling in intestinal epithelium represses canonical WNT signaling to restrict expression of WNT target genes to stem or progenitor cells; however, the mechanism remains unclear. The Hedgehog signal is transduced to GLI family transcription factors though Patched receptor, Smoothened signal transducer, and other signaling components, such as KIF27, KIF7, STK36, SUFU, and DZIP1. Here, we searched for the GLI-binding site within the promoter region of genes encoding secreted-type WNT signal inhibitors, including SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, DKK1, DKK2, DKK3, DKK4, and WIF1. The GLI-binding site was identified within the human SFRP1 promoter based on bioinformatics and human intelligence. The chimpanzee SFRP1 gene was identified within the NW_110515.1 genome sequence. The GLI-binding site of the human SFRP1 promoter was conserved in chimpanzee SFRP1, mouse Sfrp1, and rat Sfrp1 promoters. SFRP1 is the evolutionarily conserved target of the Hedgehog-GLI signaling pathway. Expression domain analyses based on text mining revealed that Indian Hedgehog (IHH), SFRP1, and WNT6 are expressed in differentiated intestinal epithelial cells, mesenchymal cells, and stem/progenitor cells, respectively. Hedgehog is secreted from differentiated epithelial cells to induce SFRP1 expression in mesenchymal cells, which keeps differentiated epithelial cells away from the effects of canonical WNT signaling. These facts indicate that SFRP1 is the Hedgehog target to confine canonical WNT signaling within stem or progenitor cells. Therefore, epigenetic CpG hypermethylation of the SFRP1 promoter during chronic persistent inflammation and aging leads to the occurrence of gastrointestinal cancers, such as colorectal cancer and gastric cancer, through the breakdown of Hedgehog-dependent WNT signal inhibition.

Hedgehog signaling pathway and gastric cancer.

Hedgehog, WNT, FGF and BMP signaling pathways network together during embryogenesis, tissue regeneration, and carcinogenesis. Aberrant activation of Hedgehog signaling pathway leads to pathological consequences in a variety of human tumors, such as gastric cancer and pancreatic cancer. Endoscopic mucosal resection (EMR), endoscopic submucosal dissection (ESD), surgical gastrectomy and chemotherapy are therapeutic options for gastric cancer; however, prognosis of advanced gastric cancer patient is still poor. Here, Hedgehog signaling pathway in human gastric cancer and its clinical applications will be reviewed. Human SHH, IHH, DHH (Hedgehog homologs), HHAT (Hedgehog acyltransferase), HHIP (Hedgehog-interacting protein), DISP1, DISP2, DISP3 (Dispatched homologs), PTCH1, PTCH2 (Patched homologs), SMO (Smoothened homolog), KIF27, KIF7 (Costal-2 homologs), STK36 (Fused homolog), SUFU (SuFu homolog), DZIP1 (Iguana homolog), GLI1, GLI2 and GLI3 (Cubitus interruptus homologs) are implicated in the Hedgehog signaling. PTCH1, FOXM1 and CCND2 are direct transcriptional targets of Hedgehog signaling. Hedgehog signaling activation leads to cell proliferation through cell cycle regulation. SHH regulates growth and differentiation within gastric mucosa through autocrine loop and FOXL1-mediated epithelial-mesenchymal interaction. SHH is implicated in stem/progenitor cell restitution of damaged gastric mucosa during chronic infection with Helicobacter pylori. SHH up-regulation, IHH upregulation and HHIP down-regulation lead to aberrant activation of Hedgehog signaling through PTCH1 to GLI1 in gastric cancer. Small molecule compounds targeted to SMO (KADD-cyclopamine, SANT1-4, Cur61414) as well as humanized anti-SHH antibodies are potent anti-cancer drugs for gastric cancer. Cocktail of Hedgehog inhibitors would be developed as novel therapeutics for gastric cancer. Single nucleotide polymorphism (SNP) and copy number polymorphism (CNP) of Hedgehog signaling genes would be utilized for genetic screening of gastric cancer, while cDNA-PCR, microarray and ELISA detecting aberrant Hedgehog signaling activation would be utilized for therapeutic optional choice. Genetic screening and precise selection of therapeutic options would contribute to the realization of personalized medicine.