Frizzled-4 Research Articles

Identification of Gene Mutations in Atypical Retinopathy of Prematurity Cases.

Purpose We have observed that some preterm infants whose fundus appears very similar to eyes with familial exudative vitreoretinopathy (FEVR) present with atypical retinopathy of prematurity (ROP). To establish a definitive diagnosis and explore the possible genetic mechanism of atypical ROP, we performed gene sequencing of these cases using next-generation sequencing technology. Methods A retrospective review of infants who presented with atypical ROP from October 2013 to February 2017 was performed. The data included gender, gestational age at birth, birth weight, family history, systemic disorders, and age-appropriate ophthalmic examinations. Fundus fluorescein angiography (FFA) of the parents was also performed. Peripheral blood was collected from the patients and their parents to sequence genes. Gene mutations were analysed. Results Genetic testing revealed that 9 infants had FEVR-related disease-causing gene mutations. Nine gene mutations were detected; 5 had already been reported, and the other 4 were novel. In the 18 eyes of these 9 patients, 9 eyes exhibited severe ROP. 5 cases had a positive family history. Conclusions Gene mutations of low-density-lipoprotein receptor-related protein 5(LRP5), frizzled-4(FZD4), Norrie disease protein (NDP), and tetraspanin-12(TSPAN12) may play a role in the pathogenesis of ROP and cause atypical ROP or preterm FEVR. The fundus lesions of ROP patients with disease-causing gene mutations were more serious. ROP cases should be carefully differentiated from preterm FEVR cases.

Mesenchymal stem cell-derived extracellular vesicles suppress the fibroblast proliferation by downregulating FZD6 expression in fibroblasts via micrRNA-29b-3p in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF), a progressive and fatal lung disease, usually leads to an irreversible distortion of the pulmonary structure. The functional roles of bone marrow-derived mesenchymal stem cells (BMSC)-secreted extracellular vesicles (EVs) in fibroblasts have been implicated, yet their actions in the treatment of IPF are not fully understood. This study investigated the roles of BMSC-derived EVs expressing miR-29b-3p in fibroblasts in IPF treatment. EVs derived from BMSCs were successfully isolated and could be internalized by pulmonary fibroblasts, and Cell Counting Kit-8 (CCK-8) and Transwell assay results identified that EVs inhibited the activation of fibroblast in IPF. miR-29b-3p, frizzled 6 (FZD6), α-skeletal muscle actin (α-SMA), and Collagen I expressions were examined, which revealed that miR-29b-3p was poorly expressed and FZD6, α-SMA, and Collagen I were overexpressed in pulmonary tissues. Dual-luciferase reporter assay results demonstrated that miR-29b-3p could inversely target FZD6 expression. The gain- and loss-of-function assays were conducted to determine regulatory effects of FZD6 and miR-29b-3p on IPF. CCK-8 and Transwell assays results displayed that BMSCs-derived EVs overexpressing miR-29b-3p contributed to inhibited pulmonary interstitial fibroblast proliferation, migration, invasion, and differentiation. Furthermore, the effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression were assessed in vivo, which confirmed the repressive effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression. Collectively, BMSCs-derived EVs overexpressing miR-29b-3p relieve IPF through FZD6.

First Report of a Known Pathogenic Variant in the FZD6 Gene, in an Iranian Family with Recessive Nail Dysplasia: A Case Re-port

Congenital Nail abnormalities are rare ectodermal defects. Autosomal recessive nail dysplasia is much rarer. Recently it has been recognized as a condition resulting in nail dystrophy in the absence of other cutaneous or extracutaneous disorders. Few case reports have identified mutations in the Frizzled 6 (FZD6) gene in families presenting with abnormal nails consistent with Non-Syndromic Congenital Nail Dysplasia. We report a family presenting, they lived in Namin a country of the Ardabil Province, northwestern Iran in 2016, for the first time in Iran in whom we identified mutations in FZD6 with abnormal nails formation.

The expression of miR-129-5p and its target genes in the skin of goats

Coat color is one of the major quality traits of animals, and miR-129-5p acts as an important regulator for melanin biosynthesis in mammals. In this study, real-time PCR and western blotting were used to examine the expression of miR-129-5p and its targets genes in the skin of different coat color goats. The results showed that the expression of miR-129-5p in the skin samples of Inner Mongolia cashmere goats (IMCG) was higher than that of Dazu black goat (DBG). Also, the target genes (tyrosinase (TYR), frizzled 6 (FZD6) and glycogen synthase kinase 3β (GSK3β)) of miR-129-5p was highly expressed in the skin samples of DBG. The expression of miR-129-5p firstly increased and then decreased with age in F1 hybrid generation of DBG and IMCG. In addition, the expression of TYR decreased with age, while the expression of MITF increased with age but then decreased. The expression of FZD6 and GSK3β in the skin samples of F1 of different ages were irregular. Our results indicated that miR-129-5p mainly affects the formation of coat color of goats by decreasing the expression of TYR. This study suggests that miR-129-5p can act as a suppressor in the formation of coat color to lay the foundation for studying the effect of miR-129-5p on melanin synthesis.

Dynamic Evolution of the Cthrc1 Genes, a Newly Defined Collagen-Like Family.

Collagen triple helix repeat containing protein 1 (Cthrc1) is a secreted glycoprotein reported to regulate collagen deposition and to be linked to the Transforming growth factor β/Bone morphogenetic protein and the Wnt/planar cell polarity pathways. It was first identified as being induced upon injury to rat arteries and was found to be highly expressed in multiple human cancer types. Here, we explore the phylogenetic and evolutionary trends of this metazoan gene family, previously studied only in vertebrates. We identify Cthrc1 orthologs in two distant cnidarian species, the sea anemone Nematostella vectensis and the hydrozoan Clytia hemisphaerica, both of which harbor multiple copies of this gene. We find that Cthrc1 clade-specific diversification occurred multiple times in cnidarians as well as in most metazoan clades where we detected this gene. Many other groups, such as arthropods and nematodes, have entirely lost this gene family. Most vertebrates display a single highly conserved gene, and we show that the sequence evolutionary rate of Cthrc1 drastically decreased within the gnathostome lineage. Interestingly, this reduction coincided with the origin of its conserved upstream neighboring gene, Frizzled 6 (FZD6), which in mice has been shown to functionally interact with Cthrc1. Structural modeling methods further reveal that the yet uncharacterized C-terminal domain of Cthrc1 is similar in structure to the globular C1q superfamily domain, also found in the C-termini of collagens VIII and X. Thus, our studies show that the Cthrc1 genes are a collagen-like family with a variable short collagen triple helix domain and a highly conserved C-terminal domain structure resembling the C1q family.

MiR-144-3p Inhibits BMSC Proliferation and Osteogenic Differentiation Via Targeting FZD4 in Steroid-Associated Osteonecrosis.

MicroRNAs have recently been recognized to be engaged in the development of bone diseases. This study was performed to elucidate the effects of miR-144-3p on proliferation and osteogenesis of mesenchymal stem cells (MSCs) from the patients with steroid-associated osteonecrosis (ONFH) and its related mechanism. The expression level of miR-144-3p in the MSCs from the proximal femur of the patients was examined by Real-time PCR. The cell proliferation ability was assayed by MTT. The differentiation ability of MSCs was assayed by Alizarin Red S (ARS) staining. The interaction between miR-144-3p and frizzled4 (FZD4) was investigated by Real-time PCR, western blot and luciferase reporter assay. ONFH samples had the obviously high expression of miR-144-3p compared to the control. MiR-144-3p had a negative effect on the proliferation and osteogenesis of MSCs. Via targeting FZD4, miR-144-3p decreased β-catenin nuclear translocation, the transcription of RUNX2 and COL1A1. Over-expression of FZD4 partially reversed miR-144-3p-induced decrease in the proliferation and osteogenesis of MSCs. MiR-144-3p might play an important role in the development of ONFH and might be used as a novel class of therapeutic targets for this disease.

The interaction of Wnt signaling members with growth factors in cultured granulosa cells.

Wnt family members have recently been distinguished in the adult ovary with potential roles in ovarian function. Though particular growth factors interact with Wnt signaling members in extraovarian cell types, it is unclear whether this interaction is applicable in the granulosa cells. Therefore, the current study aimed to determine the effect of insulin-like growth factor-1 (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-β) on Wnt ligands WNT2 and WNT4 and Wnt receptor Frizzled-4 (FZD4) protein levels in cultured mouse granulosa cells. Granulosa cells were isolated from antral follicles of adult Balb/C mice and cultured for 24 hours in the presence of 100 ng/mL of IGF-I, or EGF or FGF-β. WNT2, WNT4 and FZD4 protein levels were evaluated through western blotting after the culture process. IGF-I treated granulosa cells had significantly the highest level of WNT2 and WNT4 as well as FZD4 when compared to FGF-β and EGF groups. FGF-β group had a significantly higher level of WNT2, WNT4 and FZD4 expression when compared to EGF group. FZD4 expression was at the highest level in the IGF-I group and this difference was statistically significant for all groups including uncultured cells and vehicle group. In addition, FGF-β was shown to positively affect the adhesion of granulosa cells. This study demonstrates that IGF-I, FGF-β and EGF have differential effects on the expressions of WNT2, WNT4, and FZD4 in cultured mouse granulosa cells, suggesting that particular growth factors related to ovarian function might conduct their roles in the ovary through Wnt signaling.

A novel FZD6 mutation revealed the cause of cleft lip and/or palate in a Chinese family

Cleft lip and/or palate (CL/P) is a most common craniofacial birth defect which has multifactorial etiology. In our study, we aimed to discover the underlying etiological gene variation in a Chinese family diagnosed as non-syndromic CL/P (NSCL/P). The blood sample of the proband and her parents were detected by whole exome sequencing. The Mendelian inheritance pattern, allele frequency, variation location, function analysis and literature search were applied to filtrate and screen the mutation. Besides, the candidates were confirmed by Sanger sequencing. We meanwhile explored the conservative analysis and protein homology simulation. As a result, a start-lost mutation c.1A > GAtg/Gtg in the Frizzled-6 (FZD6) gene predicting p.Met1 was detected. The variation has not been reported before and was predicted to be harmful. The alteration caused missing of two starting amino acids that are evolutionarily conserved for FZD6 protein. Moreover, the specific structure of the mutant protein obviously changed according to the results of the homologous model. In conclusion, the results suggest c.1A > GAtg/Gtg in the FZD6 (NM_001164616) might be the genetic etiology for non-syndromic CL/P in this pedigree. Furthermore, this finding provided new etiologic information, supplementing the evidence that FZD6 is a strong potential gene for CL/P.

RETRACTED: Overexpressed lncRNA GATA6-AS1 Inhibits LNM and EMT via FZD4 through the Wnt/β-Catenin Signaling Pathway in GC

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Accumulating evidence reveals the significance of long non-coding RNAs (lncRNAs) in various cancers. The current study aimed to evaluate the role of GATA6 antisense RNA 1 (GATA6-AS1) in the epithelial-mesenchymal transition (EMT) and lymph node metastasis (LNM) in GC. GC-related microarray datasets were initially retrieved from the GEO with differentially expressed lncRNAs screened, followed by evaluation of the regulatory relationship between Frizzled 4 (FZD4) and GATA6-AS1. The detailed regulatory mechanism by which GATA6-AS1 influences the Wnt/β-catenin signaling pathway and GC cell biological behaviors was investigated by treating SGC7901 cells with overexpressed GATA6-AS1, specific antisense oligonucleotide against GATA6-AS1, and lithium chloride (LiCl; activator of the Wnt/β-catenin signaling pathway). Finally, xenograft nude mice were used to assay tumor growth and LNM in vivo. GATA6-AS1 was poorly expressed, but FZD4 was highly expressed in GC tissues and cells. Elevated GATA6-AS1 reduced FZD4 expression by recruiting enhancer of zeste homolog 2 (EZH2) and trimethylation at lysine 27 of histone H3 (H3K27me3) to the FZD4 promoter region via the inactivated Wnt/β-catenin signaling pathway, whereby cell invasion, migration, and proliferation, tumor growth, and LNM in nude mice were reduced. Taken together, overexpressed GATA6-AS1 downregulated the expression of FZD4 to inactivate the Wnt/β-catenin signaling pathway, which ultimately inhibited GC progression.

Secreted Wnt6 mediates diabetes-associated centrosome amplification via its receptor FZD4.

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of β-catenin. The treatment also activated β-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or β-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-β-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of β-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.

AGR3 promotes the stemness of colorectal cancer via modulating Wnt/β-catenin signalling

Cancer cells with stem cell properties have been acknowledged to be responsible for cancer initiation and progression. Wnt/β-catenin signalling is a major signal pathway promoting the stemness of cancer cells. Anterior gradient 3 (AGR3), a member of the protein disulfide isomerase (PDI) family, was found to be overexpressed in several cancers. However, the roles and mechanisms of AGR3 in colorectal cancer (CRC) have not been previously described. In our study, we find that AGR3 is highly expressed in CRC and associated with poor prognosis. Functional studies show that AGR3 promotes the stemness of CRC cells. Mechanically, AGR3 activates Wnt/β-catenin signalling and promotes the nuclear translocation of β-catenin to upregulate stemness related genes. Wnt/β-catenin signalling inhibition counteracts the promoting effect of AGR3 on cancer stemness. Moreover, the effect of AGR3 on Wnt/β-catenin signalling and cancer stemness depends on the presence of frizzled 4 (FZD4). Thus, our study first uncovers the stemness-promoting role and the oncogenic mechanism of AGR3 in CRC, which might provide a novel target for designing anti-CRC strategies.

Abstract 2628: Non-canonical Wnt signaling in late-stage PDAC

Abstract Pancreatic cancer has a disproportionate death-to-incidence rate largely resultant from late stage diagnoses, leading to advanced cancers which have metastasized and have increased resistance to common chemotherapeutics. This malignant progression is characterized by the increase of many molecular markers, and developing novel therapeutics targeting these late stage markers is essential for improving patient prognoses. We have identified several proteins within the non-canonical Wnt pathway that are upregulated in advanced pancreatic ductal adenocarcinomas (PDAC), and we are exploring the mechanisms driving upregulation and the resulting alterations to cell signaling. Analysis of microarray data from human derived organoids (from 6 normal pancreas samples and 38 tumor samples) revealed that two receptors in the Wnt pathway, Frizzled 2 (FZD2) and Frizzled 6 (FZD6), have increased expression levels in late-stage PDAC. The temporal increase of these non-canonical, planar cell polarity altering receptors suggests a previously underappreciated role for Wnt signaling in the progression of pancreatic cancer. The increase of these two receptors in tumor tissue was confirmed through analysis of RNA-sequencing of patient samples, including 179 tumor and 171 normal samples. Interestingly, after analyzing patient overall survival and progression free survival we found that, out of the ten members of the Frizzled receptor family, higher levels of FZD2 and FZD6 result in the worst patient prognoses. Additionally, retrospective analysis of gene expression data from Moffitt et al. suggests that FZD2 and FZD6 are higher in the aggressive “basal-like” sub-category of pancreatic cancer. To begin to understand the mechanism for these changes we have used a panel of PDAC cell lines, grown in both traditional and 3-dimensional culture. We have found that decreasing levels of FZD2 and FZD6, through stable sh-RNA knockdown, results in cells which are less motile (observed via Transwell and scratch assays) and are also less capable of anchorage independent growth. Also, cell lines with increased levels of FZD2 result in cells migrating through 3D-matrix as small cell clusters rather than as single cells, which has also been observed in partial-epithelial to mesenchymal transition cell lines. Taken together, non-canonical Wnt signaling in PDAC, mediated by FZD2 and/or FZD6 receptors, results in phenotypic alterations which are consistent with those often observed during cancer progression. These changes may help explain why patients with increased expression of FZD2 and FZD6 have worse prognoses and may also warrant more exploration into specific Frizzled inhibitors. Citation Format: Payton D. Stevens, Adam Racette, Rochelle L. Tiedemann, Bart O. Williams. Non-canonical Wnt signaling in late-stage PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2628.

Abstract 4671: Novel biological roles of the atypical WNT ligand, Norrin, on glioblastoma stem cells segregate with ASCL1 expression

Abstract Norrin is an atypical WNT ligand that binds the Frizzled-4 (FZD4) and Low-density lipoprotein receptor-related protein (LRP5/6) receptor complex to activate canonical WNT/ β-catenin signaling. Norrin/FZD4 signaling is involved in the regulation of vasculature in several tissues including the retina and for blood-brain barrier function. The role of Norrin in cancer is not very well characterized. Here, we show that NDP, the gene encoding for Norrin, is expressed in a wide range of cancer types, with a particular enrichment in glioblastoma (GBM) and lower grade glioma (LGG). Kaplan-Meier survival analysis of publicly available datasets revealed a significant correlation between NDP expression and survival in GBM, LGG and neuroblastoma. To investigate the function of NDP in GBM, we performed a set of NDP and FZD4 gain and loss of function experiments in patient-derived GBM stem cell (GNS) lines. Recently Achaete-scute homolog 1 (ASCL1) expression was shown to stratify GNS lines into two cohorts with different tumorigenic, proliferation and differentiation dynamics. Surprisingly, we found that NDP manipulation resulted in opposite effects in ASCL1hi versus ASCL1lo lines. NDP inhibited proliferation and sphere formation in ASCL1lo lines through the WNT/ β-catenin pathway, while it stimulated proliferation and sphere formation in ASCL1hi lines independently of the WNT/β-catenin pathway. The NDP/FZD4 effects on growth in both GNS subtypes were recapitulated in xenografts, confirming effects of this pathway on tumor progression in vivo. To gain insight into the cellular and molecular effects of NDP/FZD4 on GNS growth we performed immunocytochemistry (ICC) and RNA-Seq analyses in GNS cells with NDP knockdown. In parallel with our in vitro and in vivo observations, we found that NDP/FZD4 signaling affects GNS proliferation index and cell cycle kinetics and the expression of cell cycle regulatory genes in both GNS subtypes, but in opposite ways. RNA-Seq analysis identified cell cycle regulatory genes and gene sets that are commonly affected by NDP in both GNS types. Interestingly, the analysis also identified several genes and genesets unique to each GNS type, consistent with the divergence of NDP functions between GNS subtypes. Collectively, our results uncover novel functions of NDP in regulating GBM stem cell progression, and that NDP functions in GBM stem cells stratify with ASCL1 expression. Additionally, our results highlight the significance of precision medicine in targeting tumor subtypes based on the molecular signature. Citation Format: Ahmed A. Elsehemy, Hayden Selvadurai, Arturo Ortin-Martinez, Nenad Pokrajac, Yasin Mamatjan, Katherine Rowland, Kenneth Aldape, Peter Dirks, Valerie Wallace. Novel biological roles of the atypical WNT ligand, Norrin, on glioblastoma stem cells segregate with ASCL1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4671.

Optical Coherence Tomography Angiography in Familial Exudative Vitreoretinopathy: Clinical Features and Phenotype-Genotype Correlation

To evaluate the microstructure of the fovea in patients with familial exudative vitreoretinopathy (FEVR) compared to healthy controls using optical coherence tomography angiography (OCTA). In this consecutive, cross-sectional, observational case series, 41 eyes of 41 patients diagnosed as FEVR and 37 eyes in 37 control subjects were studied. OCTA was utilized to automatically measure the foveal avascular zone (FAZ) and the vessel density (VD). Inner retinal thicknesses (IRT) and central retinal thickness (CRT) were measured with the instrument caliper. Targeted next-generation sequencing was performed, and phenotype-genotype association was analyzed. Small FAZ was found in 31.70% (13/41) FEVR eyes but not in controls. Greater CRT and lower superficial foveal VD were noted in FEVR patients. FAZ is negatively correlated with IRT. Persistence of the inner retinal layer (IRL) in fovea was present in 48.78% (20/41) FEVR eyes but not found in controls. Zero percent (0/10) of patients with the low-density lipoprotein receptor-related protein 5 (LRP5) mutation, 50% (1/2) with the frizzled-4 (FZD4) mutation, and 66.67% (3/4) with the tetraspanin-12 (TSPAN12) mutation had preserved foveal IRL and small FAZ. Our data indicate FEVR status is associated with a significantly smaller FAZ, decreased vascular density in both the superficial and deep layers of parafoveal area, a thicker fovea, and an abnormally preserved IRL in fovea. In addition, patients with the LRP5 mutation had a milder phenotype than those with the FDZ4 or TSPAN12 mutations. These novel findings could provide insight into the understanding of the pathogenesis of FEVR.

Mechanically stimulated osteocytes reduce the bone-metastatic potential of breast cancer cells in vitro by signaling through endothelial cells.

Bone metastases occur in 65% to 75% of patients with advanced breast cancer and significantly worsen their survival and quality of life. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced the transendothelial migration of breast cancer cells. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. In this study, we investigated the specific effects of flow-stimulated osteocytes on the interaction between endothelial cells and breast cancer cells in vitro. We observed that CM from flow-stimulated osteocytes reduced endothelial permeability by 15% and breast cancer cell adhesion onto endothelial monolayers by 18%. The difference in adhesion was abolished with anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing antibodies. Furthermore, CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered the gene expression in bone-metastatic breast cancer cells, as shown by RNA sequencing. Specifically, breast cancer cell expression of matrix metallopeptidase 9 (MMP-9) was downregulated by 62%, and frizzled-4 (FZD4) by 61%, when the osteocytes were stimulated with flow. The invasion of these breast cancer cells across Matrigel was also reduced by 47%, and this difference was abolished by MMP-9 inhibitors. In conclusion, we demonstrated that flow-stimulated osteocytes downregulate the bone-metastatic potential of breast cancer cells by signaling through endothelial cells. This provides insights into the capability of bone mechanical regulation in preventing bone metastases; and may assist in prescribing exercise or bone-loading regimens to patients with breast cancers.

Endothelial Cell-Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature.

Objective- Blood-CNS (central nervous system) barrier defects are implicated in retinopathies, neurodegenerative diseases, stroke, and epilepsy, yet, the pathological mechanisms downstream of barrier defects remain incompletely understood. Blood-retina barrier (BRB) formation and retinal angiogenesis require β-catenin signaling induced by the ligand norrin (NDP [Norrie disease protein]), the receptor FZD4 (frizzled 4), coreceptor LRP5 (low-density lipoprotein receptor-like protein 5), and the tetraspanin TSPAN12 (tetraspanin 12). Impaired NDP/FZD4 signaling causes familial exudative vitreoretinopathy, which may lead to blindness. This study seeked to define cell type-specific functions of TSPAN12 in the retina. Approach and Results- A loxP-flanked Tspan12 allele was generated and recombined in endothelial cells using a tamoxifen-inducible Cdh5-CreERT2 driver. Resulting phenotypes were documented using confocal microscopy. RNA-Seq, histopathologic analysis, and electroretinogram were performed on retinas of aged mice. We show that TSPAN12 functions in endothelial cells to promote vascular morphogenesis and BRB formation in developing mice and BRB maintenance in adult mice. Early loss of TSPAN12 in endothelial cells causes lack of intraretinal capillaries and increased VE-cadherin (CDH5 [cadherin5 aka VE-cadherin]) expression, consistent with premature vascular quiescence. Late loss of TSPAN12 strongly impairs BRB maintenance without affecting vascular morphogenesis, pericyte coverage, or perfusion. Long-term BRB defects are associated with immunoglobulin extravasation, complement deposition, cystoid edema, and impaired b-wave in electroretinograms. RNA-sequencing reveals transcriptional responses to the perturbation of the BRB, including genes involved in vascular basement membrane alterations in diabetic retinopathy. Conclusions- This study establishes mice with late endothelial cell-specific loss of Tspan12 as a model to study pathological consequences of BRB impairment in an otherwise intact vasculature.

1266 The role of planar cell polarity gene FZD6 in melanoma progression

Melanoma is the malignant tumor of melanin-producing melanocytes in the skin. It is the leading cause of skin cancer deaths. Despite recent progress in melanoma therapeutics, the prognosis remains very poor due to the low response rate to drugs and the rapid distant metastasis in patients. A broader understanding of the biology of melanoma, especially how tumor cells metastasize, is needed to develop effective treatments for this devastating disease. The planar cell polarity (PCP) pathway controls tissue polarity during development by regulating the directional movement of cells and coordinating neighboring cells to the body/tissue axes. Increasing evidence suggests that it also plays active roles in cancer by promoting tumor cell migration and invasion. Although limited information is available regarding PCP pathway in certain cancers, its involvement in melanoma development and progression has not been studied. In our preliminary studies, we found that Frizzled6 (FZD6), one of the core PCP genes, is overexpressed in multiple human melanoma cell lines. SK-MEL28 has the highest level (over 90-fold compared to normal adult human epidermal melanocytes). To determine the role of FZD6 in melanoma cells, we made FZD6 knockout SK-MEL28 melanoma cell lines using CRISPR/Cas9. We found that knockout of FZD6 significantly reduces the migration ability of melanoma cells (using in vitro wound healing assays), while tumor cell proliferation is not affected. Current studies are underway to determine the role of FZD6 in melanoma metastasis using genetically engineered mouse models. We are also investigating the downstream mechanisms of FZD6 using both candidate approaches and non-biased transcriptome analysis. Our study will not only provide insights into the role of FZD6 in melanoma progression but also set the stage for additional studies to identify the role of other PCP genes in melanoma. The information generated from these studies will substantially broaden our knowledge on the biology of melanoma and might lead to novel therapeutic approaches for melanoma.

Downregulation of miR-3127-5p promotes epithelial-mesenchymal transition via FZD4 regulation of Wnt/β-catenin signaling in non-small-cell lung cancer.

MiR-3127-5p has been implicated as a tumor-suppressive microRNA (miRNA) in non-small-cell lung cancer (NSCLC) and its expression was associated with tumor recurrence and poor prognosis. The aim of this study was to determine whether miR-3127-5p regulates epithelial-mesenchymal transition (EMT) in NSCLC, and to investigate the underlying mechanisms. Using qRT-PCR, we examined the expression levels of miR-3127-5p in a cohort of primary NSCLC specimens with and without distant metastasis. We further performed a series of in vitro and in vivo experiments to investigate the effects and underlying mechanism of miR-3127-5p on EMT, cell migration, invasion, and adhesion in NSCLC. We found that metastatic NSCLC tissues showed markedly downregulated miR-3127-5p expression. Transforming growth factor-β1 (TGF-β1) treatment induced EMT in A549 and H1299 cells, and downregulation of miR-3127-5p could result in the similar effect. Mechanically, we demonstrated that frizzled-4 (FZD4) is a target gene and miR-3127-5p exerts its effects by regulating the Wnt/β-catenin signaling. In addition, the expression levels of FZD4 and miR-3127-5p were also negatively associated in both clinical and xenografted tumors. Overall, these findings suggest that downregulation of miR-3127-5p promotes EMT through activating the Wnt/FZD4/β-catenin signaling pathway and may represent a therapeutic target for NSCLC metastasis.

Inhibitory effects of microRNA-375 on biological behaviour of human retinal capillary endothelial cells induced by hypoxia

Background Studies showed that microRNA (miR)-375 suppresses the growth, apoptosis, migration and adhesion of tumor cells, and it plays a regulation to the changes of vascular endothelial growth factor (VEGF) in tumor tissue to arrest neovascularization.However, whether miR-375 intervenes the formation of new blood vessel in eyes is unelucidated. Objective This study was to explore the effects of miR-375 on human retinal capillary endothelial cells (HRCECs) function induced by hypoxia. Methods HRCECs were cultured using IMDM and divided into normal control group, CoCl2 model group, CoCl2+ miR-375 mimic group, CoCl2+ miR-375 mimic control group, CoCl2+ miR-375 inhibitor group and CoCl2+ miR-375 inhibitor control group, and hypoxia cell models were created by adding 200 μmol/L CoCl2.MiR-375 and frizzled 4 (FZD4) small interfering RNA (siRNA) were transfected into the cells by 50 nmol/L miRNA lipidosome for 48 hours.The proliferation of the cells was detected by MTT assay; migrated number of the cells was examined by Transwell chamber assay; ELISA was employed to detect the concentrations of VEGF and VE-cadherin in the medium; the expression of β-catenin, cyclinD1, matrix metalloproteinase-2 (MMP2) and VEGF proteins were analyzed by Western blot; tube length of vessel formation was evaluated by Matrigel assay.Cultured cells were divided into normal control group, CoCl2 model group, CoCl2+ mock group and CoCl2+ FZD4 siRNA group, the relative expression of FZD4, a miR-375 targeted gene, was detected by luciferase reporter. Results The relative expression of miR-375 mRNA was significantly increased in the CoCl2+ miR-375 mimic group compared with the CoCl2+ miR-375 mimic control group and reduced in the CoCo2+ miR-375 inhibitor group compared with the CoCo2+ miR-375 inhibitor control group (t=-19.237, 8.764, both at P<0.01), with a higher transfected efficacy for miR-375.The cell proliferative fold, migrated cell number, VEGF and VE-Cadherin contents in the medium and the tube length were significantly different among the CoCl2 model group, CoCl2+ miR-375 mimic group, CoCl2+ miR-375 mimic control group, CoCl2+ miR-375 inhibitor group and CoCl2+ miR-375 inhibitor control group (F=24.324, 26.776, 14.113, 19.225, 15.040, all at P<0.001), and those in the CoCl2+ miR-375 mimic group were evidently reduced in the CoCl2+ miR-375 mimic group compared with the CoCl2+ miR-375 mimic control group, while those in the CoCl2+ miR-375 inhibitor group were considerably elevated in comparison with the CoCl2+ miR-375 inhibitor control group (all at P<0.01). The expressions of β-catenin, cyclinD1, MMP2 and VEGF protein were significantly different among the normal control group, CoCl2 model group, CoCl2+ miR-375 mimic group, CoCl2+ miR-375 mimic control group, CoCl2+ miR-375 inhibitor group and CoCl2+ miR-375 inhibitor control group (F=11.753, 13.283, 16.770, 10.334, all at P<0.001). In addition, the cell proliferative fold, migrated cell number and the tube length were significantly increased in the CoCl2 model group and CoCl2+ mock group, and those in the CoCl2+ FZD4 siRNA group were decreased in comparison with the CoCl2+ mock group (all at P<0.05). Conclusions MiR-375 inhibits the growth, migration and tube formation ability of HRCECs in hypoxic status probably by regulating the activation of Wnt pathway via directly targeting FZD4. Key words: MicroRNAs/physiology; Retina; Endothelium, vascular; Neovascularization, pathologic; Signal transduction/physiology; Wnt proteins/metabolism; Cell proliferation /drug effects; Gene expression regulation

Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling

G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors. They can exist and act as dimers, but the requirement of dimers for agonist-induced signal initiation and structural dynamics remains largely unknown. Frizzled 6 (FZD6) is a member of Class F GPCRs, which bind WNT proteins to initiate signaling. Here, we show that FZD6 dimerizes and that the dimer interface of FZD6 is formed by the transmembrane α-helices four and five. Most importantly, we present the agonist-induced dissociation/re-association of a GPCR dimer through the use of live cell imaging techniques. Further analysis of a dimerization-impaired FZD6 mutant indicates that dimer dissociation is an integral part of FZD6 signaling to extracellular signal-regulated kinases1/2. The discovery of agonist-dependent dynamics of dimers as an intrinsic process of receptor activation extends our understanding of Class F and other dimerizing GPCRs, offering novel targets for dimer-interfering small molecules.