Claudin 1, 4, 6 and 18 isoform 2 as targets for the treatment of cancer (Review).

The 24 claudin (CLDN) genes in the human genome encode 26 representative CLDN family proteins. CLDNs are tetraspan­transmembrane proteins at tight junctions. Because several CLDN isoforms, such as CLDN6 and CLDN18.2, are specifically upregulated in human cancer, CLDN­targeting monoclonal antibodies (mAbs), antibody­drug conjugates (ADCs), bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR) T cells have been developed. In the present review, CLDN1­, 4­, 6­ and 18.2­targeting investigational drugs in clinical trials are discussed. CLDN18.2­directed therapy for patients with gastric and other types of cancer is the most advanced area in this field. The mouse/human chimeric anti­CLDN18.2 mAb zolbetuximab has a single­agent objective response rate (ORR) of 9%, and increases progression­free and overall survival in combination with chemotherapy. The human/humanized anti­CLDN18.2 mAb osemitamab, and ADCs AZD0901, IBI343 and LM­302, with single­agent ORRs of 28­60%, have been tested in phase III clinical trials. In addition, bsAbs, CAR T cells and their derivatives targeting CLDN4, 6 or 18.2 are in phase I and/or II clinical trials. AZD0901, IBI343, zolbetuximab and the anti­CLDN1 mAb ALE.C04 have been granted fast track designation or priority review designation by the US Food and Drug Administration.

FGFR-targeted therapeutics: clinical activity, mechanisms of resistance and new directions.

Fibroblast growth factor (FGF) signalling via FGF receptors (FGFR1-4) orchestrates fetal development and contributes to tissue and whole-body homeostasis, but can also promote tumorigenesis. Various agents, including pan-FGFR inhibitors (erdafitinib and futibatinib), FGFR1/2/3 inhibitors (infigratinib and pemigatinib), as well as a range of more-specific agents, have been developed and several have entered clinical use. Erdafitinib is approved for patients with urothelial carcinoma harbouring FGFR2/3 alterations, and futibatinib and pemigatinib are approved for patients with cholangiocarcinoma harbouring FGFR2 fusions and/or rearrangements. Clinical benefit from these agents is in part limited by hyperphosphataemia owing to off-target inhibition of FGFR1 as well as the emergence of resistance mutations in FGFR genes, activation of bypass signalling pathways, concurrent TP53 alterations and possibly epithelial-mesenchymal transition-related isoform switching. The next generation of small-molecule inhibitors, such as lirafugratinib and LOXO-435, and the FGFR2-specific antibody bemarituzumab are expected to have a reduced risk of hyperphosphataemia and the ability to overcome certain resistance mutations. In this Review, we describe the development and current clinical role of FGFR inhibitors and provide perspective on future research directions including expansion of the therapeutic indications for use of FGFR inhibitors, combination of these agents with immune-checkpoint inhibitors and the application of novel technologies, such as artificial intelligence.

WNT signaling and cancer stemness.

Cancer stemness, defined as the self-renewal and tumor-initiation potential of cancer stem cells (CSCs), is a cancer biology property featuring activation of CSC signaling networks. Canonical WNT signaling through Frizzled and LRP5/6 receptors is transmitted to the ß-catenin-TCF/LEF-dependent transcription machinery to up-regulate MYC, CCND1, LGR5, SNAI1, IFNG, CCL28, CD274 (PD-L1) and other target genes. Canonical WNT signaling causes expansion of rapidly cycling CSCs and modulates both immune surveillance and immune tolerance. In contrast, noncanonical WNT signaling through Frizzled or the ROR1/2 receptors is transmitted to phospholipase C, Rac1 and RhoA to control transcriptional outputs mediated by NFAT, AP-1 and YAP-TEAD, respectively. Noncanonical WNT signaling supports maintenance of slowly cycling, quiescent or dormant CSCs and promotes epithelial-mesenchymal transition via crosstalk with TGFß (transforming growth factor-ß) signaling cascades, while the TGFß signaling network induces immune evasion. The WNT signaling network orchestrates the functions of cancer-associated fibroblasts, endothelial cells and immune cells in the tumor microenvironment and fine-tunes stemness in human cancers, such as breast, colorectal, gastric and lung cancers. Here, WNT-related cancer stemness features, including proliferation/dormancy plasticity, epithelial-mesenchymal plasticity and immune-landscape plasticity, will be discussed. Porcupine inhibitors, ß-catenin protein-protein interaction inhibitors, ß-catenin proteolysis targeting chimeras, ROR1 inhibitors and ROR1-targeted biologics are investigational drugs targeting WNT signaling cascades. Mechanisms of cancer plasticity regulated by the WNT signaling network are promising targets for therapeutic intervention; however, further understanding of context-dependent reprogramming trajectories might be necessary to optimize the clinical benefits of WNT-targeted monotherapy and applied combination therapy for patients with cancer.

Precision medicine for human cancers with Notch signaling dysregulation (Review).

NOTCH1, NOTCH2, NOTCH3 and NOTCH4 are transmembrane receptors that transduce juxtacrine signals of the delta­like canonical Notch ligand (DLL)1, DLL3, DLL4, jagged canonical Notch ligand (JAG)1 and JAG2. Canonical Notch signaling activates the transcription of BMI1 proto­oncogene polycomb ring finger, cyclin D1, CD44, cyclin dependent kinase inhibitor 1A, hes family bHLH transcription factor 1, hes related family bHLH transcription factor with YRPW motif 1, MYC, NOTCH3, RE1 silencing transcription factor and transcription factor 7 in a cellular context­dependent manner, while non­canonical Notch signaling activates NF­κB and Rac family small GTPase 1. Notch signaling is aberrantly activated in breast cancer, non­small­cell lung cancer and hematological malignancies, such as T­cell acute lymphoblastic leukemia and diffuse large B­cell lymphoma. However, Notch signaling is inactivated in small­cell lung cancer and squamous cell carcinomas. Loss­of­function NOTCH1 mutations are early events during esophageal tumorigenesis, whereas gain­of­function NOTCH1 mutations are late events during T­cell leukemogenesis and B­cell lymphomagenesis. Notch signaling cascades crosstalk with fibroblast growth factor and WNT signaling cascades in the tumor microenvironment to maintain cancer stem cells and remodel the tumor microenvironment. The Notch signaling network exerts oncogenic and tumor­suppressive effects in a cancer stage­ or (sub)type­dependent manner. Small­molecule γ­secretase inhibitors (AL101, MRK­560, nirogacestat and others) and antibody­based biologics targeting Notch ligands or receptors [ABT­165, AMG 119, rovalpituzumab tesirine (Rova­T) and others] have been developed as investigational drugs. The DLL3­targeting antibody­drug conjugate (ADC) Rova­T, and DLL3­targeting chimeric antigen receptor­modified T cells (CAR­Ts), AMG 119, are promising anti­cancer therapeutics, as are other ADCs or CAR­Ts targeting tumor necrosis factor receptor superfamily member 17, CD19, CD22, CD30, CD79B, CD205, Claudin 18.2, fibroblast growth factor receptor (FGFR)2, FGFR3, receptor­type tyrosine­protein kinase FLT3, HER2, hepatocyte growth factor receptor, NECTIN4, inactive tyrosine­protein kinase 7, inactive tyrosine­protein kinase transmembrane receptor ROR1 and tumor­associated calcium signal transducer 2. ADCs and CAR­Ts could alter the therapeutic framework for refractory cancers, especially diffuse­type gastric cancer, ovarian cancer and pancreatic cancer with peritoneal dissemination. Phase III clinical trials of Rova­T for patients with small­cell lung cancer and a phase III clinical trial of nirogacestat for patients with desmoid tumors are ongoing. Integration of human intelligence, cognitive computing and explainable artificial intelligence is necessary to construct a Notch­related knowledge­base and optimize Notch­targeted therapy for patients with cancer.

Molecular genetics and targeted therapy of WNT-related human diseases (Review).

Canonical WNT signaling through Frizzled and LRP5/6 receptors is transduced to the WNT/ß-catenin and WNT/stabilization of proteins (STOP) signaling cascades to regulate cell fate and proliferation, whereas non-canonical WNT signaling through Frizzled or ROR receptors is transduced to the WNT/planar cell polarity (PCP), WNT/G protein-coupled receptor (GPCR) and WNT/receptor tyrosine kinase (RTK) signaling cascades to regulate cytoskeletal dynamics and directional cell movement. WNT/ß-catenin signaling cascade crosstalks with RTK/SRK and GPCR-cAMP-PKA signaling cascades to regulate ß-catenin phosphorylation and ß-catenin-dependent transcription. Germline mutations in WNT signaling molecules cause hereditary colorectal cancer, bone diseases, exudative vitreoretinopathy, intellectual disability syndrome and PCP-related diseases. APC or CTNNB1 mutations in colorectal, endometrial and prostate cancers activate the WNT/ß-catenin signaling cascade. RNF43, ZNRF3, RSPO2 or RSPO3 alterations in breast, colorectal, gastric, pancreatic and other cancers activate the WNT/ß-catenin, WNT/STOP and other WNT signaling cascades. ROR1 upregulation in B-cell leukemia and solid tumors and ROR2 upregulation in melanoma induce invasion, metastasis and therapeutic resistance through Rho-ROCK, Rac-JNK, PI3K-AKT and YAP signaling activation. WNT signaling in cancer, stromal and immune cells dynamically orchestrate immune evasion and antitumor immunity in a cell context-dependent manner. Porcupine (PORCN), RSPO3, WNT2B, FZD5, FZD10, ROR1, tankyrase and ß-catenin are targets of anti-WNT signaling therapy, and ETC-159, LGK974, OMP-18R5 (vantictumab), OMP-54F28 (ipafricept), OMP-131R10 (rosmantuzumab), PRI-724 and UC-961 (cirmtuzumab) are in clinical trials for cancer patients. Different classes of anti-WNT signaling therapeutics are necessary for the treatment of APC/CTNNB1-, RNF43/ZNRF3/RSPO2/RSPO3- and ROR1-types of human cancers. By contrast, Dickkopf-related protein 1 (DKK1), SOST and glycogen synthase kinase 3ß (GSK3ß) are targets of pro-WNT signaling therapy, and anti-DKK1 (BHQ880 and DKN-01) and anti-SOST (blosozumab, BPS804 and romosozumab) monoclonal antibodies are being tested in clinical trials for cancer patients and osteoporotic post-menopausal women. WNT-targeting therapeutics have also been applied as reagents for in vitro stem-cell processing in the field of regenerative medicine.

Cancer genetics and genomics of human FOX family genes.

Forkhead-box (FOX) family proteins, involved in cell growth and differentiation as well as embryogenesis and longevity, are DNA-binding proteins regulating transcription and DNA repair. The focus of this review is on the mechanisms of FOX-related human carcinogenesis. FOXA1 is overexpressed as a result of gene amplification in lung cancer, esophageal cancer, ER-positive breast cancer and anaplastic thyroid cancer and is point-mutated in prostate cancer. FOXA1 overexpression in breast cancer and prostate cancer is associated with good or poor prognosis, respectively. Single nucleotide polymorphism (SNP) within the 5'-UTR of the FOXE1 (TTF2) gene is associated with thyroid cancer risk. FOXF1 overexpression in breast cancer is associated with epithelial-to-mesenchymal transition (EMT). FOXM1 is overexpressed owing to gene amplification in basal-type breast cancer and diffuse large B-cell lymphoma (DLBCL), and it is transcriptionally upregulated owing to Hedgehog-GLI, hypoxia-HIF1α or YAP-TEAD signaling activation. FOXM1 overexpression leads to malignant phenotypes by directly upregulating CCNB1, AURKB, MYC and SKP2 and indirectly upregulating ZEB1 and ZEB2 via miR-200b downregulation. Tumor suppressor functions of FOXO transcription factors are lost in cancer cells as a result of chromosomal translocation, deletion, miRNA-mediated repression, AKT-mediated cytoplasmic sequestration or ubiquitination-mediated proteasomal degradation. FOXP1 is upregulated as a result of gene fusion or amplification in DLBCL and MALT lymphoma and also repression of miRNAs, such as miR-1, miR-34a and miR-504. FOXP1 overexpression is associated with poor prognosis in DLBCL, gastric MALT lymphoma and hepatocellular carcinoma but with good prognosis in breast cancer. In neuroblastoma, the entire coding region of the FOXR1 (FOXN5) gene is fused to the MLL or the PAFAH1B gene owing to interstitial deletions. FOXR1 fusion genes function as oncogenes that repress transcription of FOXO target genes. Whole-genome sequencing data from tens of thousands of human cancers will uncover the mutational landscape of FOX family genes themselves as well as FOX-binding sites, which will be ultimately applied for cancer diagnostics, prognostics, and therapeutics.

Integrative genomic analyses of CXCR4: transcriptional regulation of CXCR4 based on TGFbeta, Nodal, Activin signaling and POU5F1, FOXA2, FOXC2, FOXH1, SOX17, and GFI1 transcription factors.

CXCR4, CD133, CD44 and ABCG2 are representative transmembrane proteins expressed on the surfaces of normal and/or cancer stem cells. CXCR4 is co-expressed with POU5F1 in endodermal precursors and adult-tissue stem cells. CXCR4 is expressed in a variety of human tumors, such as breast cancer, prostate cancer, pancreatic cancer, and gastric cancer. CXCR4 is a G protein-coupled receptor (GPCR) for CXCL12 (SDF1) chemokine, and the CXCL12-CXCR4 signaling axis is involved in proliferation, survival, migration, and homing of cancer cells. Integrative genomic analyses of CXCR4 gene were carried out to elucidate the mechanisms of CXCR4 expression in stem cells, because CXCR4 is a key molecule occupying the crossroads of oncology, immunology, gerontology and regenerative medicine. Human CXCR4 promoter region with binding sites for HIF1alpha, ETS1, NF-kappaB and GLI was not conserved in mouse and rat Cxcr4 orthologs. Proximal enhancer region with palindromic Smad-binding sites, FOX-binding site, POU-binding site, triple SOX17-binding sites, bHLH-binding site, TCF/LEF-binding site, and double GFI1-binding sites was almost completely conserved among human, chimpanzee, mouse, and rat CXCR4 orthologs. TGFbeta, Nodal, and Activin signals induce CXCR4 upregulation based on Smad2/3 and FOX family members, such as FOXA2, FOXC2, and FOXH1. CXCR4 is expressed in endodermal precursors due to the existence of triple SOX17-binding sites around the POU-binding site instead of the POU5F1-SOX2 joint motif. Because CXCR4 is downregulated by p53-GFI1 signaling axis, p53 mutation in cancer stem cells leads to CXCR4 upregulation. CXCR4 is also upregulated by TGFbeta and Hedgehog signals in tumor cells at the invasion front. Small molecule compound or human antibody targeted to CXCR4 will be applied for cancer therapeutics focusing on cancer stem cells at the primary lesion as well as metastasis or recurrence niches, such as bone marrow and peritoneal cavity.

Integrative genomic analyses of WNT11: transcriptional mechanisms based on canonical WNT signals and GATA transcription factors signaling.

We and others previously cloned and characterized vertebrate WNT11 orthologs, which are involved in gastrulation, neurulation, cardiogenesis, nephrogenesis, and chondrogenesis during fetal development. WNT11 orthologs activate both canonical and non-canonical WNT signaling cascades depending on the expression profile of WNT receptors, such as Frizzled family members, LRP6, ROR2, and RYK. Human WNT11 is expressed in breast cancer, gastric cancer, esophageal cancer, colorectal cancer, neuroblastoma, Ewing sarcoma, and prostate cancer. Canonical WNT signals and GATA family members are involved in WNT11 transcription during embryogenesis of model animals; however, precise mechanisms of WNT11 expression remain unclear. Here, refined integrative genomic analyses of WNT11 are carried out to elucidate the mechanisms of WNT11 transcription. The WNT11 gene was found to encode two isoforms by using alternative first exons. WNT11 isoform A (NM_004626.2 RefSeq) consists of exons 2, 3, 4, 5 and 6, whereas WNT11 isoform B consists of exons 1, 2, 3, 4, 5 and 6. We identified double TCF/LEF-binding sites within the proximal promoter regions -48-bp position from the TSS of human WNT11 isoform B and -43-bp position from the TSS of human WNT11 isoform A), and also double GATA-binding sites within intron 2 of human WNT11 gene (+933-bp and +5001-bp positions from TSS of human WNT11 isoform A). Double TCF/LEF- and double GATA-binding sites within the regulatory regions of human WNT11 gene were conserved in other mammalian WNT11 orthologs. These facts indicate that canonical WNT signals and GATA family members directly upregulate WNT11 transcription. Canonical WNT-induced WNT11 activates non-canonical WNT signaling cascades to induce cellular movement, and also activates the Ca2+-MAP3K7-NLK signaling cascade to break the canonical WNT signaling. Canonical WNT-to-WNT11 signaling loop is involved in cellular migration during embryogenesis as well as tumor invasion during carcinogenesis.

Integrative genomic analyses of ZEB2: Transcriptional regulation of ZEB2 based on SMADs, ETS1, HIF1alpha, POU/OCT, and NF-kappaB.

Epithelial-to-mesenchymal transition (EMT) is defined as phenotypic change of epithelial cells into mesenchymal cells. EMT, allowing cellular dissociation from epithelial tissues, plays a key role in invasion and metastasis during carcinogenesis as well as in gastrulation and neurulation during embryogenesis. SNAI1/Snail, SNAI2/Slug, ZEB1/deltaEF1/ZFHX1A, ZEB2/SIP1/ZFHX1B, TWIST1/TWIST, and TWIST2/DERMO1 are representative EMT regulators. ZEB2 represses transcription of CDH1, CLDN4, CCND1, TERT, SFRP1, ALPL and miR-200b-200a-429 primary miRNA, and upregulates transcription of mesenchymal markers. ZEB2 is relatively highly expressed in brain corpus callosum and monocytes. ZEB2 is expressed in various types of human tumors, such as breast cancer, gastric cancer, and pancreatic cancer. TGFbeta, TNFalpha, IL1, AKT and hypoxia signals are involved in ZEB2 upregulation and EMT induction; however precise mechanisms of ZEB2 transcription remained unclear. Here, refined integrative genomic analyses of ZEB2 gene were carried out. ZEB2 was co-expressed with POU3F2 (BRN2) and POU3F3 (BRN1) in brain corpus callosum, spinal cord, and fetal brain, whereas ZEB2 was co-expressed with POU2F2 (OCT2) in monocytes. Ets-Smad-binding CGGAGAC motif, bHLH-binding site, and POU/OCT-binding site within proximal promoter region, and NF-kappaB-binding site within intron 2 were completely conserved in human ZEB2, chimpanzee ZEB2, cow ZEB2, mouse Zeb2, rat Zeb2, and chicken zeb2 genes. In addition, HIF1alpha-binding site within proximal promoter region was conserved in mammalian ZEB2 orthologs. Consensus binding site for Hedgehog effector GLI was not identified within or adjacent to the 7-kb regions of human ZEB2 gene. TGFbeta, TNFalpha, IL1, and hypoxia signals directly upregulate ZEB2 to induce EMT, growth arrest, and senescence, whereas Hedgehog signals indirectly upregulate ZEB2 via TGFbeta. Together these facts indicate that ZEB2, occupying the crossroads of inflammation, aging and carcinogenesis, is an important target for drug discovery.

Transcriptional mechanisms of WNT5A based on NF-kappaB, Hedgehog, TGFbeta, and Notch signaling cascades.

WNT5A is a cancer-associated gene involved in invasion and metastasis of melanoma, breast cancer, pancreatic cancer, and gastric cancer. WNT5A transduces signals through Frizzled, ROR1, ROR2 or RYK receptors to beta-catenin-TCF/LEF, DVL-RhoA-ROCK, DVL-RhoB-Rab4, DVL-Rac-JNK, DVL-aPKC, Calcineurin-NFAT, MAP3K7-NLK, MAP3K7-NF-kappaB, and DAG-PKC signaling cascades in a context-dependent manner. SNAI1 (Snail), CD44, G3BP2, and YAP1 are WNT5A target genes. We and other groups previously reported that IL6- or LIF-induced signaling through JAK-STAT3 signaling cascade is involved in WNT5A upregulation (STAT3-WNT5A signaling loop). Here, refined integrative genomic analyses of WNT5A were carried out to elucidate other mechanisms of WNT5A transcription. The WNT5A gene was found to encode two isoforms by using alternative first exons 1A and 1B. Quadruple Smad-binding elements (SBEs), single Sp1-binding site (GC-box), PPARgamma-binding site, C/EBP-binding site and bHLH-binding site within the promoter A region, 5'-adjacent to exon 1A, were conserved in human WNT5A, chimpanzee WNT5A, mouse Wnt5a, and rat Wnt5a. NF-kappaB-binding site, CUX1-binding site, double SBEs and double GC-boxes within the promoter B region, 5'-adjacent to exon 1B, were conserved in mammalian WNT5A orthologs. Quadruple FOX-binding sites and double SBEs within ultra-conserved intron 1 were also conserved in mammalian WNT5A orthologs. Conserved NF-kappaB-binding site within the WNT5A promoter B region elucidated the mechanisms that TNFalpha and toll-like receptor (TLR) signals upregulate WNT5A via MAP3K7. Quadruple FOX-binding sites rather than GLI-binding site revealed that Hedgehog signals induce WNT5A upregulation indirectly via FOX family members, such as FOXA2, FOXC2, FOXE1, FOXF1 and FOXL1. TGFbeta signals were found to upregulate WNT5A expression directly through the Smad complex, and also indirectly through Smad-induced CUX1 and MAP3K7-mediated NF-kappaB. Together these facts indicate that WNT5A is transcribed based on multiple mechanisms, such as NF-kappaB, Hedgehog, TGFbeta, and Notch signaling cascades.

Transcriptional regulation of WNT2B based on the balance of Hedgehog, Notch, BMP and WNT signals.

We cloned and characterized human WNT2B in 1996, and then others cloned and characterized mouse, chicken, and zebrafish WNT2B orthologs. WNT2B is expressed in several types of human cancer, such as basal cell carcinoma, gastric cancer, breast cancer, head/neck squamous cell carcinoma, cervical cancer and leukemia. WNT2B is one of canonical WNTs transducing signals through Frizzled (FZD) and LRP5/LRP6 receptors to beta-catenin-TCF/LEF signaling cascade. Here, refined integrative genomic analyses on WNT2B orthologs were carried out to elucidate its transcriptional mechanisms. GLI-, double FOX-, HES/HEY-, bHLH-, and Sp1-binding sites within mammalian WNT2B promoter were well conserved. Because GLI1, FOXA2, FOXC2, FOXE1, FOXF1 and FOXL1 are direct target genes of Hedgehog-GLI2 signaling cascade, Hedgehog signals should induce WNT2B upregulation through GLI family members as well as FOX family members. Notch, BMP and Hedgehog signals inhibit WNT2B expression via HES/HEY-binding to N-box, whereas BMP and WNT signals inhibit bHLH transcription factor-induced WNT2B expression via ID1, ID2, ID3, MSX1 or MSX2. Together these facts indicate that Hedgehog signals and bHLH transcription factors are involved in WNT2B upregulation, which is counteracted by BMP, WNT and Notch signals. Mesenchymal BMP induces IHH expression in gastrointestinal epithelial cells, and then epithelial Hedgehog induces WNT2B and BMP4 expression in mesenchymal cells. NF-kappaB signals induce SHH upregulation, and WNT2B is upregulated in inflammatory bowel disease (IBD). BMP-IHH and inflammation-SHH signaling loops are involved in WNT2B up-regulation during embryogenesis, adult tissue homeostasis, and carcinogenesis.

Comparative integromics on non-canonical WNT or planar cell polarity signaling molecules: transcriptional mechanism of PTK7 in colorectal cancer and that of SEMA6A in undifferentiated ES cells.

Non-canonical WNT and planar cell polarity (PCP) are overlapping but distinct signaling pathways, which control convergent extension, neural tube closure, orientation of cilia and sensory hair cells, axon guidance, and cell motility. Non-canonical WNT signals, regulated by the interaction of WNT, WNT antagonist, Frizzled and ROR2, are transduced to JNK, ROCK, PKC, MAP3K7, and NFAT signaling cascades. PCP signals, regulated by the interaction of VANGL-PRICKLE complex, CELSR and Frizzled-DVL complex, are transduced to JNK, ROCK, and other uncharacterized signaling cascades. PTK7 signaling, regulated by SEMA6 and Plexin-A family members, affects PCP pathway through VANGL. Here, integrative genomic analyses on WNT5A, WNT5B, WNT11, FZD3, FZD6, ROR1, ROR2, RYK, CELSR1, CELSR2, CELSR3, VANGL1, VANGL2, PRICKLE1, PRICKLE2, PTK7, SEMA6A, SEMA6B, SEMA6C and SEMA6D were carried out. PTK7 and SEMA6A were expressed in undifferentiated embryonic stem (ES) cells, SEMA6A in endodermal progenitors, CELSR1, VANGL1 and PTK7 in gastrointestinal tumors. CELSR2, PRICKLE2 and SEMA6C were expressed in fetal brain, CELSR2, PRICKLE1 and SEMA6A in adult brain, WNT5A and CELSR3 in adult brain tumors. These facts indicate class switches of non-canonical WNT or PCP signaling molecules during embryogenesis and carcinogenesis. TCF/LEF-, SP1-, and 5 bHLH-binding sites within human PTK7 promoter were conserved in chimpanzee, rhesus monkey, mouse, and rat PTK7 orthologs, which explained the mechanism of PTK7 upregulation in colorectal cancer. NANOG-, SOX2-, and POU5F1 (OCT3/OCT4)-binding sites within intron 1 of the human SEMA6A gene were conserved in chimpanzee, rhesus monkey, mouse, and rat SEMA6A orthologs, which explained the mechanism of SEMA6A upregulation in undifferentiated ES cells. Most of non-canonical WNT or PCP signaling molecules, except PTK7 and SEMA6A, were not frequently expressed in undifferentiated human ES cells. Non-canonical WNT or PCP signaling pathway, activated to orchestrate gastrulation and neurulation, was relatively downregulated in undifferentiated ES cells derived from inner cell mass of blastocysts.

Integrative genomic analyses on HES/HEY family: Notch-independent HES1, HES3 transcription in undifferentiated ES cells, and Notch-dependent HES1, HES5, HEY1, HEY2, HEYL transcription in fetal tissues, adult tissues, or cancer.

Notch signaling pathway maintains stem cells through transcriptional activation of HES/HEY family members to repress tissue-specific transcription factors. Here, comparative integromic analyses on HES/HEY family members were carried out. HES3 gene encodes two isoforms due to alternative promoters. Complete coding sequence of HES3 variant 2 was determined by curating CX755241.1 EST. Refined phylogenetic analysis using HES3 variant 2 instead of variant 1 revealed that mammalian bHLH transcription factors with Orange domain were grouped into HES subfamily (HES1, HES2, HES3, HES4, HES5, HES6, HES7) and HEY subfamily (HEY1, HEY2, HEYL, HESL/HELT, DEC1/BHLHB2, DEC2/BHLHB3). Eight amino-acid residues were added to the C-terminal WRPW motif in human HES3 due to lineage specific T to G nucleotide change at stop codon of chimpanzee, rat, and mouse HES3 orthologs. HES1 and HES3 were expressed in undifferentiated embryonic stem (ES) cells. HES1 was also expressed in fetal tissues, and regenerating liver. HES1, HEY1 and HEY2 were expressed in endothelial cells. HES1, HES4 and HES6 were expressed in gastric cancer, HES1 and DEC1 in pancreatic cancer, HES1, HES2, HES4, HES6 and DEC2 in colorectal cancer. HES6 was also expressed in other tumors, such as brain tumors, melanoma, small cell lung cancer, retinoblastoma, ovarian cancer, and breast cancer. Double NANOG-binding sites, CSL/RBPSUH-binding site and TATA-box in HES1 promoter, NANOG-, SOX2-, POU5F1/OCT3/OCT4-binding sites and TATA-box in HES3 promoter, double CSL-binding sites in HES5 promoter, SOX2-, POU-binding sites and TATA-box in HES6 promoter, and CSL-binding site in HEY1, HEY2 and HEYL promoters were evolutionarily conserved. However, double CSL-binding sites in mouse Hes7 promoter were not conserved in human HES7 promoter. Together these facts indicate that HES1 and HES3 were target genes of the ES cell-specific network of transcription factors, and that HES1, HES5, HEY1, HEY2 and HEYL were target genes of Notch signaling pathway.

WNT signaling pathway and stem cell signaling network.

WNT signals are transduced to the canonical pathway for cell fate determination, and to the noncanonical pathway for control of cell movement and tissue polarity. Canonical WNT signals are transduced through Frizzled family receptors and LRP5/LRP6 coreceptor to the beta-catenin signaling cascade. Microtubule affinity-regulating kinase (PAR-1) family kinases, casein kinase I epsilon (CKI epsilon), and FRAT are positive regulators of the canonical WNT pathway, whereas APC, AXIN1, AXIN2, CKI alpha, NKD1, NKD2, beta TRCP1, beta TRCP2, ANKRD6, Nemo-like kinase (NLK), and peroxisome proliferator-activated receptor gamma (PPAR gamma) are negative regulators. Nuclear complex, consisting of T-cell factor/lymphoid enhancer factor, beta-catenin, BCL9/BCL9L, and PYGO, activates transcription of canonical WNT target genes such as FGF20, DKK1, WISP1, MYC, CCND1, and Glucagon (GCG). Noncanonical WNT signals are transduced through Frizzled family receptors and ROR2/RYK coreceptors to the Dishevelled-dependent (Rho family GTPases and c-jun NH(2)-terminal kinase) or the Ca(2+)-dependent (NLK and nuclear factor of activated T cells) signaling cascades. WNT signals are context-dependently transduced to both pathways based on the expression profile of WNT, SFRP, WIF, DKK, Frizzled receptors, coreceptors, and the activity of intracellular WNT signaling regulators. Epigenetic silencing and loss-of-function mutation of negative regulators of the canonical WNT pathway occur in a variety of human cancer. WNT, fibroblast growth factor (FGF), Notch, Hedgehog, and transforming growth factor beta/bone morphogenetic protein signaling network are implicated in the maintenance of tissue homeostasis by regulating self-renewal of normal stem cells as well as proliferation or differentiation of progenitor (transit-amplifying) cells. Breakage of the stem cell signaling network leads to carcinogenesis. Nonsteroidal anti-inflammatory drugs and PPAR gamma agonists with the potential to inhibit the canonical WNT signaling pathway are candidate agents for chemoprevention. ZTM000990 and PKF118-310 are lead compounds targeted to the canonical WNT signaling cascade. Anti-WNT1 and anti-WNT2 monoclonal antibodies show in vitro effects in cancer treatment. After the optimization, derivatives of small-molecule compound and human monoclonal antibody targeted to the WNT signaling pathway could be used in cancer medicine.

Comparative integromics on JMJD1C gene encoding histone demethylase: conserved POU5F1 binding site elucidating mechanism of JMJD1C expression in undifferentiated ES cells and diffuse-type gastric cancer.

Epigenetic modifications of genomic DNA and histones alter the chromatin structure to regulate the accessibility of transcription factors to the promoter or enhancer regions. In 2003, we identified and characterized JMJD1C (TRIP8) consisting of TRI8H1 domain with C2HC4-type zinc finger-like motif, TRI8H2 domain with thyroid hormone receptor beta-binding region, and JmjC domain. JMJD1A (TSGA), JMJD1B (5qNCA) and JMJD1C with the common domain architecture are histone H3K9 demethylases implicated in the nuclear hormone receptor-based transcriptional regulation. Here, comparative integromics on JMJD1C gene is reported. JMJD1C variant 1, previously reported, consists of exons 1, 2 and 3-26, while JMJD1C variant 2 characterized in this study was transcribed from novel exon 1B located 5' to exon 3. Four human JMJD1C ESTs were transcribed from exon 1, while 14 human JMJD1C ESTs from exon 1B. All of 26 mouse Jmjd1c ESTs were transcribed from exon 1b. These facts indicate that JMJD1C variant 2 transcribed from exon 1B was the major transcript. Human JMJD1C variant 2 with TRI8H1, TRI8H2, and JmjC domains showed 85.7% total-amino-acid identity with mouse Jmjd1c. Human JMJD1C mRNA was expressed in undifferentiated embryonic stem (ES) cells, pancreatic islet, diffuse-type gastric cancer, and other tissues or tumors. Mouse Jmjd1c mRNA was expressed in fertilized egg, blastocyst, undifferentiated ES cells, embryonic germ cells, c-Kit+/Sca-1+/Lin- hematopoietic stem cells, pancreatic islet, and other tissues. Comparative genomics analyses revealed that binding sites for POU5F1 (OCT3/OCT4), AP-1, and bHLH transcription factors within the promoter region located 5' to exon 1B of human JMJD1C gene were conserved in chimpanzee, cow, mouse and rat JMJD1C orthologs. POU5F1-mediated expression of JMJD1C histone demethylase is implicated in the reactivation of silenced genes in undifferentiated ES cells, pancreatic islet, and diffuse-type gastric cancer.

AP1- and NF-kappaB-binding sites conserved among mammalian WNT10B orthologs elucidate the TNFalpha-WNT10B signaling loop implicated in carcinogenesis and adipogenesis.

WNT signals are context-dependently transduced to canonical and non-canonical signaling cascades. We cloned and characterized wild-type human WNT10B, while another group cloned aberrant human WNT10B with Gly60Asp amino-acid substitution. Proto-oncogene WNT10B is expressed in gastric cancer, pancreatic cancer, breast cancer, esophageal cancer, and cervical cancer. Because WNT10B blocks adipocyte differentiation, coding SNP of WNT10B gene is associated with familial obesity. In 2001, we reported WNT10B upregulation by TNFalpha. Here, comparative integromics analyses on WNT10B orthologs were performed to elucidate the transcriptional mechanism of WNT10B. Chimpanzee WNT10B and cow Wnt10b genes were identified within NW_001223159.1 and AC150975.2 genome sequences, respectively, by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee WNT10B and cow Wnt10b showed 98.7% and 95.1% total-amino-acid identity with human WNT10B, respectively. N-terminal signal peptide, 24 Cys residues, two Asn-linked glycosylation sites, and Gly60 of human WNT10B were conserved among mammalian WNT10B orthologs. Transcription start site of human WNT10B gene was 106-bp upstream of NM_003394.2 RefSeq 5'-end. Number of GC di-nucleotide repeats just down-stream of WNT10B transcription start site varied among primates and human population. Comparative genomics analyses revealed that double AP1-binding sites in the 5'-flanking promoter region and NF-kappaB-binding site in intron 3 were conserved among human, chimpanzee, cow, mouse, and rat WNT10B orthologs. Because TNFalpha signaling through TNFR1 and TRADD/RIP/TRAF2 complex activates JUN kinase (JNK) and IkappaB kinase (IKK) signaling cascades, conserved AP1- and NF-kappaB-binding sites explain the mechanism of TNFalpha-induced WNT10B upregulation. TNFalpha-WNT10B signaling loop is the negative feedback mechanism of adipogenesis to prevent obesity and metabolic syndrome. On the other hand, TNFalpha-WNT10B signaling loop is implicated in carcinogenesis. Inhibitors of TNFalpha-WNT10B signaling loop could be utilized for the prevention or treatment of cancer associated with chronic inflammation, such as gastric, liver, breast and pancreatic cancer.

Conserved POU/OCT- and GATA-binding sites in 5'-flanking promoter region of mammalian WNT8B orthologs.

WNT family members are secreted-type glycoproteins regulating cell fate, planar cell polarity, cell adhesion, and cell movement. WNT signals are context-dependently transduced to the canonical pathway for the transcriptional up-regulation of MYC, CCND1, FGF20, JAG1, WISP1 and DKK1 genes, and also to the non-canonical pathway for the activation of RHOA, JNK, PKC, NFAT and NLK signaling cascades. We cloned and characterized the wild-type human WNT8B, while another group the aberrant human WNT8B with Gly230Ala and Arg284Leu amino-acid substitutions. Although WNT8B is undetectable in normal adult tissues by using Northern blot analyses, WNT8B is expressed in gastric cancer, pancreatic cancer, colorectal cancer, breast cancer, and embryonal tumors. Here, comparative integromics on WNT8B orthologs were investigated by using bioinformatics (Techint) and human intelligence (Humint). Cow Wnt8b gene was identified within NW_001494361.1 genome sequence. Predicted sequence XM_582222.3 was an artificial cow Wnt8b with aberrant prediction for the first exon. Cow Wnt8b complete coding sequence was found to encode a 350-amino-acid protein, which showed 96.9% total-amino-acid identity with human WNT8B. Comparative proteomics revealed that N-terminal signal peptide, 22 Cys residues, two Asn-linked glycosylation sites, Gly230, and Arg284 of human WNT8B were conserved among mammalian WNT8B orthologs. Comparative genomics revealed that POU/OCT- and GATA-binding sites in the 5'-flanking promoter region were conserved among human, chimpanzee, cow, mouse, and rat WNT8B orthologs. In silico expression analyses revealed that human WNT8B was expressed in embryoid body derived from embryonic stem (ES) cells, hepatocyte progenitors derived from ES cells, fetal brain, diffuse-type gastric cancer, colorectal cancer, prostate cancer, and ovarian fibrotheoma. Based on the expression profiles of POU and GATA family transcription factors, it was revealed that WNT8B expression in hepatocyte progenitors derived from human ES cells is due to POU5F1 (OCT3/OCT4) and GATA3, and also that WNT8B expression in diffuse-type gastric cancer is due to POU5F1 and GATA6.

Comparative integromics on FZD7 orthologs: conserved binding sites for PU.1, SP1, CCAAT-box and TCF/LEF/SOX transcription factors within 5'-promoter region of mammalian FZD7 orthologs.

Canonical WNT signals are transduced through Frizzled (FZD) family receptor and LRP5/LRP6 co-receptor to upregulate MYC, CCND1, FGF20, JAG1, WISP1 and DKK1 genes, while non-canonical WNT signals are transduced through FZD family receptor and PTK7/ROR2/RYK co-receptor to activate RHOA/RHOU/RAC/CDC42, JNK, PKC, NFAT and NLK signaling cascades. FZD7, expressed in the normal gastrointestinal tract, is upregulated in esophageal cancer, gastric cancer, colorectal cancer, and hepatocellular carcinoma. Here, chimpanzee FZD7 and cow Fzd7 genes were identified and characterized by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee FZD7 and cow Fzd7 genes were identified within NW_001232110.1 and AC173037.2 genome sequences, respectively. Chimpanzee FZD7 and cow Fzd7 showed 100% and 97.2% total-amino-acid identity with human FZD7. All of the nine amino-acid residues substituted between human FZD7 and human FzE3 were identical to those of human FZD7 in chimpanzee, cow, mouse and rat FZD7 orthologs. Functional analyses using FzE3 with multiple cloning artifacts and/or sequencing errors are invalid. FZD7 orthologs were seven-transmembrane proteins with extracellular Frizzled domain, leucine zipper motif around the 5th transmembrane domain, and cytoplasmic DVL- and PDZ-binding motifs. Ser550 and Ser556 of FZD7 orthologs were putative aPKC phosphorylation sites. Dimerization and Ser550/556 phosphorylation were predicted as regulatory mechanisms for the signaling through FZD7. Transcriptional start site of human FZD7 gene was 735-bp upstream of NM_003507.1 RefSeq 5'-end. In addition to gastrointestinal cancer, hepatocellular cancer and pancreatic cancer, human FZD7 mRNAs were expressed in blastocysts, undifferentiated embryonic stem (ES) cells, ES-derived endodermal progenitors, ES-derived neural progenitors, fetal cochlea, retinal pigment epithelium, olfactory epithelium, regenerating liver, and multiple sclerosis. Comparative genomics analyses revealed that the binding sites for PU.1, SP1/Krüppel-like, CCAAT-box, and TCF/LEF/SOX transcription factors were conserved among 5'-promoter regions of mammalian FZD7 orthologs.

STAT3-induced WNT5A signaling loop in embryonic stem cells, adult normal tissues, chronic persistent inflammation, rheumatoid arthritis and cancer (Review).

Leukemia inhibitory factor (LIF), oncostatin M, leptin, ciliary neurotrophic factor, cardiotrophin 1, cardiotrophin-like cytokine factor 1, interleukin 6 (IL6), interleukin 11 and interleukin 27 activate the gp130-JAK-STAT3 signaling cascade. Here, WNT5A was characterized as the evolutionarily conserved target of the STAT3 signaling cascade based on 11-bp-spaced tandem STAT3-binding sites within intron 4 of human, chimpanzee, cow, mouse and rat WNT5A orthologs. Canonical WNT5A signaling through Frizzled and LRP5/LRP6 receptors activates FGF20, WISP1, MYC and CCND1 transcription for the maintenance of stem/progenitor cells, while non-canonical WNT5A signaling through Frizzled and ROR2/PTK7/RYK receptors activates the RHOA, JNK, NLK and NFAT signaling cascades for the control of tissue polarity, cell adhesion or movement. LIF-induced Wnt5a activates canonical Wnt signaling in mouse embryonic stem cells for self-renewal. STAT3-induced Wnt5a activates non-canonical Wnt signaling in rat cardiac myocytes for N-cadherin-dependent aggregation. IL6, secreted from epithelial cells or macrophages, induces WNT5A upregulation in mesenchymal cells. WNT5A then activates canonical WNT signaling in epithelial cells. IL6-induced WNT5A activates canonical WNT signaling for autocrine proliferation of human synovial fibroblasts in rheumatoid arthritis. IL-6 signaling is activated during human chronic atrophic gastritis with Helicobacter pylori infection, and aberrant Stat3 signaling activation gives rise to mouse gastric tumors. WNT5A is frequently upregulated in human primary gastric cancer due to tumor-stromal interaction. WNT5A might be downregulated in advanced cancer with poorer prognosis due to genetic alterations compensating WNT5A signaling. Oncogenic WNT5A activates canonical WNT signaling in cancer stem cells for self-renewal, and non-canonical WNT signaling at the tumor-stromal interface for invasion and metastasis. SNP of genes encoding components of the cytokine-induced WNT5A signaling loop is a predicted risk factor for RA and cancer, especially diffuse-type gastric and pancreatic cancer. Humanized anti-IL6 receptor antibody and WNT5A mimetic small-molecule antagonist could be applied to personalized medicine for RA and cancer driven by the IL6-induced WNT5A signaling loop.