Growth Arrest Specific 1 Research Articles

TCF7L1 regulates colorectal cancer cell migration by repressing GAS1 expression

Dysregulated Wnt/β-catenin signaling is a common feature of colorectal cancer (CRC). The T-cell factor/lymphoid enhancer factor (TCF/LEF; hereafter, TCF) family of transcription factors are critical regulators of Wnt/β-catenin target gene expression. Of the four TCF family members, TCF7L1 predominantly functions as a transcriptional repressor. Although TCF7L1 has been ascribed an oncogenic role in CRC, only a few target genes whose expression it regulates have been characterized in this cancer. Through transcriptome analyses of TCF7L1 regulated genes, we noted enrichment for those associated with cellular migration. By silencing and overexpressing TCF7L1 in CRC cell lines, we demonstrated that TCF7L1 promoted migration, invasion, and adhesion. We localized TCF7L1 binding across the CRC genome and overlapped enriched regions with transcriptome data to identify candidate target genes. The growth arrest-specific 1 (GAS1) gene was among these and we demonstrated that GAS1 is a critical mediator of TCF7L1-dependent CRC cell migratory phenotypes. Together, these findings uncover a novel role for TCF7L1 in repressing GAS1 expression to enhance migration and invasion of CRC cells.

Abstract 4728: Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal cancer

Abstract Background: Cetuximab, an epidermal growth factor receptor (EGFR)-targeting IgG1 monoclonal antibody (mAb), results in beneficial, yet limited, clinical improvement in metastatic colorectal cancer (mCRC) patients with wild-type (WT) KRAS gene. Numerous studies have shown that circRNAs are aberrantly expressed in tumors and play crucial roles in tumor growth and metastasis in various human malignancies, including colorectal cancer (CRC). One such circRNA, circ-EGFR, has been identified as having associations with tumor progression and chemotherapy response in diverse cancer types. However, exploring the functional role and underlying mechanisms of circ-EGFR in the context of the response to EGFR-targeted therapy may offer new therapeutic avenues for mCRC. Methods: The expression level of circ-EGFR (hsa_circ_0080229) was evaluated in cetuximab-sensitive and resistant RAS and BRAF WT CRC cell lines by using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The biological function of circ-EGFR was investigated by loss- and gain-of-function assays in cultured cells and an animal model. The potential targets of circ-EGFR were identified through in-silico analysis, and the circ-EGFR/miR-942-3p/GAS1 axis pathway was verified using the luciferase reporter assay and RT-qPCR. The association of circ-EGFR expression with clinical prognosis in mCRC patients with different cetuximab responses was analyzed by Kaplan-Meier survival analysis. Results: The expression of circ-EGFR was remarkedly higher in sensitive (DiFi and SW48) compared with resistant cell lines (CaCO2 and SNU-C1) after exposure of cells to cetuximab (sensitive vs. resistant: p < 0.001). Biologically, genetic depletion of circ-EGFR significantly enhanced cell proliferation, migration [DiFi: sh-circ-EGFR vs. shNC: Foldchange (FC) = 1.97, p < 0.001; SW48: FC = 1.96, p < 0.001], and invasion (DiFi: sh-circ-EGFR vs. shNC: FC = 2.23, p < 0.001; SW48: FC = 1.78, p < 0.001) after 24-48 hours of cetuximab treatment in DiFi and SW48 cell lines, and its overexpression suppressed tumor growth in vivo under cetuximab treatment. Mechanically, we identified circ-EGFR as a sponge for miR-942-3p to regulate the expression of Growth Arrest Specific 1 (GAS1), which promoted sensitivity to cetuximab treatment in CRC. Notably, our findings identified circ-EGFR as a promising therapeutic indicator in clinical applications. Conclusion: Our study illustrated that circ-EGFR plays a vital role in affecting cetuximab efficacy by regulating the miR-942-3p/GAS1 axis in CRC. This holds promise for improving patient selection and management in this malignancy. Citation Format: Silei Sui, Caiming Xu, Yuan Li, Wenyi Wei, Joan Maurel, Man Li, Ajay Goel. Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4728.

Bifidobacterium adolescentis orchestrates CD143+ cancer-associated fibroblasts to suppress colorectal tumorigenesis by Wnt signaling-regulated GAS1.

The interplay between gut microbiota and tumor microenvironment (TME) in the pathogenesis of colorectal cancer (CRC) is not well explored. Here, we elucidated the functional role of Bifidobacterium adolescentis (B.a) on CRC and investigated its possible mechanism on the manipulation of cancer-associated fibroblasts (CAFs) in CRC. Different CRC animal models and various cell line models were established to explore the function of B.a on CRC. The single-cell RNA sequencing (scRNA-seq) or flow cytometry was used to detect the cell subsets in the TME of CRC. Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), or immunofluorescence staining were performed to examine the activation of Wnt signaling and growth arrest specific 1 (GAS1) on CD143+ CAFs. Chromatin immunoprecipitation quantitative real-time PCR (CHIP-qPCR) was performed to investigate the regulation of transcription factor 4 (TCF4) on GAS1. Multi-immunofluorescence assay examined the expression level of CD143 and GAS1 on tissue microarray. We found that B.a abundance was significantly reduced in CRC patients from two independent cohorts and the bacteria database of GMrepo. Supplementation with B.a suppressed ApcMin/+ spontaneous or AOM/DSS-induced tumorigenesis in mice. scRNA-seq revealed that B.a facilitated a subset of CD143+ CAFs by inhibiting the infiltration of Th2 cells, while promoting the TNF-alpha+ B cells in TME. CD143+ CAFs highly expressed GAS1 and exhibited tumor suppressive effect. Mechanistically, GAS1 was activated by the Wnt/β-catenin signaling in CD143+ CAFs. B.a abundance was correlated with the expression level of CD143 and GAS1. The level of CD143+ CAFs predicted the better survival outcome in CRC patients. These results highlighted that B.a induced a new subset of CD143+ CAFs by Wnt signaling-regulated GAS1 to suppress tumorigenesis and provided a novel therapeutic target for probiotic-based modulation of TME in CRC.

Low-scale production and purification of a biologically active optimized form of the antitumor protein growth arrest specific 1 (GAS1) in a mammalian system for post-translational analysis

Growth arrest specific 1 (GAS1) is a protein involved in cell arrest that induces quiescence and apoptosis, when it is overexpressed in tumor cells. We have previously reported the generation of a soluble truncated protein known as tGAS1, with antitumoral capacity. In this paper two lentiviral constructs were developed, containing: the native human gene sequence fused to a Hisx6-tag (tG1Hc); or a synthetic and optimized DNA sequence (tG1Hs). Both transgenes encode for a version of the human tGAS1 protein with the addition of a Hisx6-tag in the C-terminus (tG1H). Resultant VSV-G pseudotyped lentiviruses were used to generate HEK-293FT and U87-MG cell cultures stable for the constitutive production of tG1H proteins. The data show that only U87-MG expressed the recombinant proteins when infected. In fact, conditioned medium obtained from U87-MG cells producing tG1Hs mRNA preserves anti-proliferative and pro-apoptotic properties. Noteworthy, viral particles carrying the tG1Hs transgene inhibit tumor growth in a heterotopic xenograft mice model. Additionally, the recombinant tG1H protein was purified by multiple affinity protocols and used to perform further experiments to analyze post-translational processing or affinity co-precipitation protocols. In the present work we have studied the N-glycosylation of GAS1 in the purified protein.

GAS1 Promotes Ferroptosis of Liver Cells in Acetaminophen-Induced Acute Liver Failure.

Purpose: Acute liver failure (ALF) is a clinically fatal disease that leads to the rapid loss of normal liver function. Acetaminophen (APAP) is a leading cause of drug-induced ALF. Ferroptosis, defined as iron-dependent cell death associated with lipid peroxide accumulation, has been shown to be strongly associated with APAP-induced liver injury. Growth arrest-specific 1 (GAS1) is a growth arrest-specific gene, which is closely related to the inhibition of cell growth and promotion of apoptosis. However, the functional role and underlying mechanism of GAS1 in APAP-induced ferroptosis remain unknown. Methods: We established liver-specific overexpression of GAS1 (GAS1AAV8-OE) mice and the control (GAS1AAV8-vector) mice by tail vein injection of male mice with adeno-associated virus. APAP at 500 mg/kg was intraperitoneally injected into these two groups of mice to induce acute liver failure. The shRNA packaged by the lentivirus inhibits GAS1 gene expression in human hepatoma cell line HepaRG (HepaRG-shNC and HepaRG-shGAS1-2) and primary hepatocytes of mice with liver-specific overexpression of GAS1 were isolated and induced by APAP in vitro to further investigate the regulatory role of GAS1 in APAP-induced acute liver failure. Results: APAP-induced upregulation of ferroptosis, levels of lipid peroxides and reactive oxygen species, and depletion of glutathione were effectively alleviated by the ferroptosis inhibitor, ferrostatin-1, and downregulation of GAS1 expression. GAS1 overexpression promoted ferroptosis-induced lipid peroxide accumulation via p53, inhibiting its downstream target, solute carrier family 7 member 11. Conclusion: Collectively, our findings suggest that GAS1 overexpression plays a key role in aggravating APAP-induced acute liver injury by promoting ferroptosis-induced accumulation of lipid peroxides.

A positive feedback loop between TAZ and miR-942-3p modulates proliferation, angiogenesis, epithelial-mesenchymal transition process, glycometabolism and ROS homeostasis in human bladder cancer

BackgroundTranscriptional coactivator with PDZ-binding motif (TAZ) has been reported to be involved in tumor progression, angiogenesis, epithelial-mesenchymal transition (EMT), glycometabolic modulation and reactive oxygen species (ROS) buildup. Herein, the underlying molecular mechanisms of the TAZ-induced biological effects in bladder cancer were discovered.MethodsqRT-PCR, western blotting and immunohistochemistry were performed to determine the levels of TAZ in bladder cancer cells and tissues. CCK-8, colony formation, tube formation, wound healing and Transwell assays and flow cytometry were used to evaluate the biological functions of TAZ, miR-942-3p and growth arrest-specific 1 (GAS1). QRT-PCR and western blotting were used to determine the expression levels of related genes. Chromatin immunoprecipitation and a dual-luciferase reporter assay were performed to confirm the interaction between TAZ and miR-942. In vivo tumorigenesis and colorimetric glycolytic assays were also conducted.ResultsWe confirmed the upregulation and vital roles of TAZ in bladder cancer. TAZ-induced upregulation of miR-942-3p expression amplified upstream signaling by inhibiting the expression of large tumor suppressor 2 (LATS2, a TAZ inhibitor). MiR-942-3p attenuated the impacts on cell proliferation, angiogenesis, EMT, glycolysis and ROS levels induced by TAZ knockdown. Furthermore, miR-942-3p restrained the expression of GAS1 to modulate biological behaviors.ConclusionOur study identified a novel positive feedback loop between TAZ and miR-942-3p that regulates biological functions in bladder cancer cells via GAS1 expression and illustrated that TAZ, miR-942-3p and GAS1 might be potential therapeutic targets for bladder cancer treatment.

Construction and prognostic analysis of miRNA-mRNA regulatory network in liver metastasis from colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most common cancers in the world, and liver metastasis is the leading cause of colorectal cancer-related deaths. However, the mechanism of liver metastasis in CRC has not been clearly elucidated.MethodsThree datasets from the Gene Expression Omnibus (GEO) database were analyzed to obtain differentially expressed genes (DEGs), which were subjected to functional enrichment analysis and protein-protein interaction analysis. Subsequently, mRNA-miRNA network was constructed, and the associated DEGs and DEMs were performed for prognostic analysis. Finally, we did infiltration analysis of growth arrest specific 1 (GAS1)-associated immune cells.ResultsWe obtained 325 DEGs and 9 differentially expressed miRNAs (DEMs) between primary CRC and liver metastases. Enrichment analysis and protein-protein interactions (PPI) further revealed the involvement of DEGs in the formation of the inflammatory microenvironment and epithelial-mesenchymal transition (EMT) during the liver metastases process in CRC. Survival analysis demonstrated that low-expressed GAS1 as well as low-expressed hsa-miR-33b-5p was a favorable prognostic indicator of overall survival. Further exploration of GAS1 revealed that its expression was interrelated with the infiltration of immune cells in tumor tissues.ConclusionsIn summary, DEGs, DEMs, and their interactions found in liver metastasis of CRC may provide a basis for further understanding of the mechanism of CRC metastasis.

The systemic administration of neural stem cells expressing an inducible and soluble form of growth arrest specific 1 inhibits mammary gland tumor growth and the formation of metastases

Background aimsMetastasis to different organs is the major cause of death in breast cancer patients. The poor clinical prognosis and lack of successful treatments for metastatic breast cancer patients demand the development of new tumor-selective therapies. Thus, it is necessary to develop treatments capable of releasing therapeutic agents to both primary tumors and metastases that avoid toxic side effects in normal tissue, and neural stem cells are an attractive vehicle for tracking tumor cells and delivering anti-cancer agents. The authorspreviously demonstrated that a soluble form of growth arrest specific 1 (GAS1) inhibits the growth of triple-negative breast tumors and glioblastoma. MethodsIn this study, the authors engineered ReNcell CX (EMD Millipore, Temecula, CA, USA) neural progenitor cells to express truncated GAS1 (tGAS1) under a tetracycline/on inducible system using lentiviral vectors. ResultsHere the authors show that treatment with ReNcell-tGAS1 in combination with tetracycline decreased primary tumor growth and inhibited the formation of metastases in tumor-bearing mice by diminishing the phosphorylation of AKT and ERK1/2 in orthotopic mammary gland tumors. Moreover, the authors observed that ReNcell-tGAS1 prolonged the survival of 4T1 tumor-bearing mice. ConclusionsThese data suggest that the delivery of tGAS1 by ReNcell cells could be an effective adjuvant for the treatment of triple-negative breast cancer.

MicroRNAs in Small Extracellular Vesicles Indicate Successful Embryo Implantation during Early Pregnancy.

Synchronous communication between the developing embryo and the receptive endometrium is crucial for embryo implantation. Thus, uterine receptivity evaluation is vital in managing recurrent implantation failure (RIF). The potential roles of small extracellular vesicle (sEV) miRNAs in pregnancy have been widely studied. However, the systematic study of sEVs derived from endometrium and its cargos during the implantation stage have not yet been reported. In this study, we isolated endometrium-derived sEVs from the mouse endometrium on D2 (pre-receptive phase), D4 (receptive phase), and D5 (implantation) of pregnancy. Herein, we reveal that multivesicular bodies (MVBs) in the endometrium increase in number during the window of implantation (WOI). Moreover, our findings indicate that CD63, a well-known sEV marker, is expressed in the luminal and glandular epithelium of mouse endometrium. The sEV miRNA expression profiles indicated that miR-34c-5p, miR-210, miR-369-5p, miR-30b, and miR-582-5p are enriched during WOI. Further, we integrated the RIF’s database analysis results and found out that miR-34c-5p regulates growth arrest specific 1 (GAS1) for normal embryo implantation. Notably, miR-34c-5p is downregulated during implantation but upregulated in sEVs. An implication of this is the possibility that sEVs miR-34c-5p could be used to evaluate uterine states. In conclusion, these findings suggest that the endometrium derived-sEV miRNAs are potential biomarkers in determining the appropriate period for embryo implantation. This study also has several important implications for future practice, including therapy of infertility.

Investigating Novel Roles for Hedgehog Co‐Receptors During Craniofacial Development

Hedgehog (HH) signaling is essential for correct patterning of vertebrate embryos, including during craniofacial development. HH ligands signal through the canonical receptor Patched1 (PTCH1), and three co‐receptors–growth arrest specific 1 (GAS1), cell adhesion molecule‐related/down‐regulated by oncogenes (CDON), and brother of CDON (BOC). Together, these co‐receptors are required during embryogenesis to mediate proper HH signaling. However, recent genetic studies suggest tissue‐specific, antagonistic roles for these proteins. For example, Gas1 restricts the HH pathway during mouse tooth development, while Boc antagonizes HH signaling during zebrafish jaw formation. Here I explored possible antagonistic roles for GAS1, CDON and BOC in HH‐dependent mammalian craniofacial development. Genetic deletion of these co‐receptors results in variable holoprosencephaly (HPE) phenotypes (failure to divide the forebrain into two cerebral hemispheres). Gas1 mutant mice display a spectrum of HPE phenotypes, and significant patterning defects in the neural tube and limb. In contrast, genetic deletion of Boc does not perturb patterning in any of these tissues. Strikingly, the combined deletion of Boc and Gas1 results in more severe neural tube and limb patterning defects than those observed in Gas1 single mutants. However, the Gas1 craniofacial phenotype is partially ameliorated in Gas1;Boc double mutant embryos as determined by the levels of NKX2.1, Gli1, Ptch1 and internasal distance quantitation. Micro‐computed tomography and skeletal analyses reveal that the rescue of the craniofacial defects in Gas1;Boc double mutants is restricted to the nasal bone and nasal capsule; other craniofacial bones are more severely affected, similar to what is observed in the neural tube and the limb. This effect does not appear to be due to differential GAS1 and BOC function in the forebrain neuroepithelium as overexpression of these co‐receptors in the developing chicken forebrain both promote HH signaling. Instead, I hypothesize that these co‐receptors have distinct, tissue‐specific functions in the craniofacial mesenchyme or the surface ectoderm. To further explore an antagonistic role for BOC during craniofacial development, I am currently deleting Boc in a sensitized HH genetic background through breeding with Ptch1 and Shh heterozygous mutant animals. These findings suggest that GAS1 and BOC do not function simply as redundant HH co‐receptors, but instead display distinct, tissue‐specific functions, particularly during craniofacial development.Support or Funding InformationNational Sciences Foundation Graduate Research Fellowship Program (DGE 1256260), Rackham Pre‐Candidate Graduate Student Research Grant, Bradley Merill Patten Fellowship and Organogenesis Training Grant (NIH T32HD007505)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Additive effects of the combined expression of soluble forms of GAS1 and PTEN inhibiting glioblastoma growth.

The overexpression of GAS1 (Growth Arrest Specific 1) in glioma cells induces cell cycle arrest and apoptosis. We previously demonstrated that the apoptotic process set off by GAS1 is caused by its capacity to inhibit the Glial cell-derived neurotrophic factor (GDNF)-mediated intracellular survival signaling pathway. Whereas on the other hand, PTEN is a tumor suppressor, inactive in many tumors, and both GAS1 and PTEN inhibit the PI3K/AKT pathway. Therefore, it is relevant to investigate the potential additive effect of the overexpression of GAS1 and PTEN on tumor growth. In particular, we employed secreted forms of both GAS1 (tGAS1) and PTEN (PTEN-LONG, or PTEN-L) and tested their combined effect on glioma cells. We observed that the co-expression of both the proteins inhibited the growth of U-87 MG human glioblastoma cells more effectively than when independently expressed, and decreased the activity of both AKT and ERK1/2. Interestingly, the combination of the soluble forms was always the most effective treatment. To improve the transfer of tGAS1 and PTEN-L, we employed a lentiviral vector with a p2A peptide-enabled dual expression system that allowed the generation of the two proteins using a single promoter (CMV), in equimolar amounts. The viral vector reduced the growth of U-87 MG cells in vitro and had a striking effect in inhibiting their proliferation after inoculating it into the immunosuppressed mice. The present results support a potential adjuvant role for the combined use of tGAS1 and PTEN-L in the treatment of glioblastoma.

The Growth arrest specific 1 (Gas1) gene is transcriptionally regulated by NeuroD1 via two distal E-boxes

Growth arrest specific 1 (GAS1) is a signaling mediator for the development of the central nervous system that works as a co-receptor for sonic hedgehog (SHH) to induce the amplification of neural progenitors during the patterning of the mammalian neural tube and establishing granular cells in the cerebellum. Recently, we confirmed that Gas1 is also expressed by neural progenitors of the developing cortex and the dentate gyrus of the hippocampus. The presence of GAS1 in progenitor stages indicates that one of its principal roles is the maintenance of these cells during neurogenic events. However, the signals responsible for the expression of Gas1 in progenitor cells are unknown, an aspect that is relevant to understand its functions during neurogenesis. Here, we focused on elucidating the mechanisms of the transcriptional regulation of Gas1 and using comparative genomics methods found two highly conserved E-boxes in the Gas1 promoter which mediate its up-regulation by NeuroD1. Additionally, we found that GAS1 and NeuroD1 co-localize in the neocortex, the dentate gyrus of the hippocampus and the external granular layer of the cerebellum, suggesting a previously unsuspected regulatory relationship. Our data indicate that Gas1 is a direct target of NeuroD1 during the induction of the neurogenic program.

Downregulation of OCLN and GAS1 in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of kidney cancer. This carcinoma is histologically characterized by the presence of clear and abundant cytoplasm. In the present study, we sought to identify genes differentially expressed in ccRCC and build a molecular profile of this cancer. We selected genes described in the literature related to cellular differentiation and proliferation. We analyzed the gene and protein expression by quantitative PCR (qPCR) and immunohistochemistry, respectively, and examined possible epigenetic mechanisms that regulate their expression in ccRCC samples and cell lines. Occludin (OCLN) and growth arrest-specific1 (GAS1) genes were underexpressed in ccRCC, and we report that miR-122 and miR-34a, respectively, may regulate their expression in this cancer. Furthermore, we showed by qPCR and immunohistochemistry that solute carrier family2 member1 (SLC2A1) was significantly overexpressed in ccRCC. The set of genes identified in the present study furthers our understanding of the molecular basis and development of ccRCC.

GAS1 is present in the cerebrospinal fluid and is expressed in the choroid plexus of the adult rat.

Growth arrest specific 1 (GAS1) is a GPI-anchored protein that inhibits proliferation when overexpressed in tumors but during development it promotes proliferation and survival of different organs and tissues. This dual ability is caused by its capacity to interact both by inhibiting the signaling induced by the glial cell line-derived neurotrophic factor and by facilitating the activity of the sonic hedgehog pathway. GAS1 is expressed as membrane bound in different organs and as a secreted form by glomerular mesangial cells. In the developing central nervous system, GAS1 is found in neural progenitors; however, it continues to be expressed in the adult brain. Here, we demonstrate that soluble GAS1 is present in the cerebrospinal fluid (CSF) and it is expressed in the choroid plexus (CP) of the adult rat, the main producer of CSF. Additionally, we confirm the presence of GAS1 in blood plasma and liver of the adult rat, the principal source of blood plasma proteins. The pattern of expression of GAS1 is perivascular in both the CP and the liver. In vitro studies show that the fibroblast cell line NIH/3T3 expresses one form of GAS1 and releases two soluble forms into the supernatant. Briefly, in the present work, we show the presence of GAS1 in adult rat body fluids focusing in the CSF and the CP, and suggest that secreted GAS1 exists as two different isoforms.

Growth Arrest Specific-1 (GAS1) Is a C/EBP Target Gene That Functions in Ovulation and Corpus Luteum Formation in Mice.

Ovulation and luteinization are initiated in preovulatory follicles by the luteinizing hormone (LH) surge; however, the signaling events that mediate LH actions in these follicles remain incompletely defined. Two key transcription factors that are targets of LH surge are C/EBPalpha and C/EBPbeta, and their depletion in granulosa cells results in complete infertility. Microarray analyses of these mutant mice revealed altered expression of a number of genes, including growth arrest specific-1 (Gas1). To investigate functions of Gas1 in ovulation- and luteinization-related processes, we crossed Cyp19a1-Cre and Gas1flox/flox mice to conditionally delete Gas1 in granulosa and cumulus cells. While expression of Gas1 is dramatically increased in granulosa and cumulus cells around 12–16 h post-human chorionic gonadotropin (hCG) stimulation in wild-type mice, this increase is abolished in Cebpa/b double mutant and in Gas1 mutant mice. GAS1 is also dynamically expressed in stromal cells of the ovary independent of C/EBPalpha/beta. Female Gas1 mutant mice are fertile, exhibit enhanced rates of ovulation, increased fertility, and higher levels of Areg and Lhcgr mRNA in granulosa cells. The morphological appearance and vascularization of corpora lutea appeared normal in these mutant females. Interestingly, levels of mRNA for a number of genes (Cyp11a1, Star, Wnt4, Prlr, Cd52, and Sema3a) associated with luteinization are decreased in corpora lutea of Gas1 mutant mice as compared with controls at 24 h post-hCG; these differences were no longer detectable by 48 h post-hCG. The C/EBP target Gas1 is induced in granulosa cells and is associated with ovulation and luteinization.

Solution structure and biophysical characterization of the multifaceted signalling effector protein growth arrest specific-1.

BackgroundThe protein growth arrest specific-1 (GAS1) was discovered based on its ability to stop the cell cycle. During development it is involved in embryonic patterning, inhibits cell proliferation and mediates cell death, and has therefore been considered as a tumor suppressor. GAS1 is known to signal through two different cell membrane receptors: Rearranged during transformation (RET), and the sonic hedgehog receptor Patched-1. Sonic Hedgehog signalling is important in stem cell renewal and RET mediated signalling in neuronal survival. Disorders in both sonic hedgehog and RET signalling are connected to cancer progression. The neuroprotective effect of RET is controlled by glial cell-derived neurotrophic factor family ligands and glial cell-derived neurotrophic factor receptor alphas (GFRαs). Human Growth arrest specific-1 is a distant homolog of the GFRαs.ResultsWe have produced and purified recombinant human GAS1 protein, and confirmed that GAS1 is a monomer in solution by static light scattering and small angle X-ray scattering analysis. The low resolution solution structure reveals that GAS1 is more elongated and flexible than the GFRαs, and the homology modelling of the individual domains show that they differ from GFRαs by lacking the amino acids for neurotrophic factor binding. In addition, GAS1 has an extended loop in the N-terminal domain that is conserved in vertebrates after the divergence of fishes and amphibians.ConclusionsWe conclude that GAS1 most likely differs from GFRαs functionally, based on comparative structural analysis, while it is able to bind the extracellular part of RET in a neurotrophic factor independent manner, although with low affinity in solution. Our structural characterization indicates that GAS1 differs from GFRα’s significantly also in its conformation, which probably reflects the functional differences between GAS1 and the GFRαs.

The role of the sonic hedgehog signalling pathway in patients with midline defects and congenital hypopituitarism.

The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH.

A soluble form of GAS1 inhibits tumor growth and angiogenesis in a triple negative breast cancer model

We previously demonstrated the capacity of GAS1 (Growth Arrest Specific 1) to inhibit the growth of gliomas by blocking the GDNF–RET signaling pathway. Here, we show that a soluble form of GAS1 (tGAS1), decreases the number of viable MDA MB 231 human breast cancer cells, acting in both autocrine and paracrine manners when secreted from producing cells. Moreover, tGAS1 inhibits the growth of tumors implanted in female nu/nu mice through a RET-independent mechanism which involves interfering with the Artemin (ARTN)-GFRα3-(GDNF Family Receptor alpha 3) mediated intracellular signaling and the activation of ERK. In addition, we observed that the presence of tGAS1 reduces the vascularization of implanted tumors, by preventing the migration of endothelial cells. The present results support a potential adjuvant role for tGAS1 in the treatment of breast cancer, by detaining tumor growth and inhibiting angiogenesis.

Neural stem cells producing an inducible and soluble form of Gas1 target and inhibit intracranial glioma growth

Background aimsGlioblastoma multiforme (GBM) is the most common and lethal primary brain tumor and current treatments have not improved its prognosis. Therefore, new strategies and therapeutic agents should be investigated. Growth arrest specific-1 (Gas1) is a protein that induces cell arrest and apoptosis of gliomas and a soluble form, tGas1, increases these effects acting in both autocrine and paracrine manners. Moreover, neural stem cells (NSCs) can be used as a vehicle to transport therapeutic molecules because they have innate tropism towards tumors. MethodsLentiviral vectors were used to obtain NSCs capable of expressing tGas1 in a regulated manner. The ability of engineered NSCs to track and reach GBM in vivo, produce tGas1, and their efficacy decreasing tumor growth and increasing the overall health and survival time of nude mice implanted with GBM were assessed. ResultsThe overexpression of tGas1 from NSCs decreased viability and induced cell arrest and apoptosis of GBM cells and also, albeit in a reduced manner, of NSCs themselves. NSCs migrate from one cerebral hemisphere to the contralateral, reach GBM, express the tGas1 transgene when induced by tetracycline and produce the protein. Tumor volume decreased by 77% compared with controls, and tGas1 improved the overall health and increased the survival time of mice implanted with GBM by 75%. ConclusionsWe demonstrated that tGas1 has an antineoplastic effect, and the results support the potential of tGas1 as an adjuvant for the treatment of gliomas.

Down-regulation of miR-34a alleviates mesangial proliferation in vitro and glomerular hypertrophy in early diabetic nephropathy mice by targeting GAS1

AimsDiabetic nephropathy (DN) is a major diabetic complication characterized by mesangial proliferation and glomerular hypertrophy. MicroRNAs might play an important role in these pathological processes. The aim of this study is to examine the possible association of miR-34a as one of the microRNAs with DN and underlying mechanisms in vitro and in vivo. MethodsAccording to previous results of microarray which compared the different microRNAs between diabetic and normal control mice, miR-34a was chosen and its expression was detected by qRT-PCR. Cell viability was then assessed using Cell Counting Kit-8 (CCK8) and 5-ethynyl-20-deoxyuridine (EDU) incorporation. Antagomir was injected in db/db mice to down regulate miR-34a. Average diameter of glomeruli was analyzed by periodic acid-Schiff (PAS) stain of kidney. Luciferase gene report assay was then performed to identify the target gene of miR-34a. Additional immunoblotting and immunohistochemical analyses were implemented to verify the expression level of growth arrest-specific 1 (GAS1). ResultsMiR-34a expression level was increased under high glucose condition in vitro and in vivo. Down-regulation of miR-34a inhibits mice mesangial cells (MMCs) proliferation in vitro and alleviates glomerular hypertrophy in vivo. GAS1 was proved to be the target of miR-34a through luciferase report. Moreover, up-regulation of GAS1 expression was observed in the presence of miR-34a antagomir as compared with miR-34a antagomir-NC in high-glucose-treated MMCs and db/db mice, respectively. ConclusionsMiR-34a regulated mesangial proliferation and glomerular hypertrophy by directly inhibiting GAS1 in early DN.