Frequently Rearranged In Advanced T-cell Lymphomas 1 Research Articles

FRAT1 promotes the angiogenic properties of human glioblastoma cells via VEGFA.

Glioblastoma is a common central nervous system tumor and despite considerable advancements in treatment patient prognosis remains poor. Angiogenesis is a significant prognostic factor in glioblastoma, anti‑angiogenic treatments represent a promising therapeutic approach. Vascular endothelial growth factor A (VEGFA) is a predominant regulator of angiogenesis and mounting evidence suggests that the Wnt signaling pathway serves a significant role in tumor angiogenesis. As a positive regulator of the Wnt/β‑catenin signaling pathway, frequently rearranged in advanced T‑cell lymphomas‑1 (FRAT1) is highly expressed in human glioblastoma and is significantly associated with glioblastoma growth, invasion and migration, as well as poor patient prognosis. Bioinformatics analysis demonstrated that both VEGFA and FRAT1 were highly expressed in most tumor tissues and associated with prognosis. However, whether and how FRAT1 is involved in angiogenesis remains to be elucidated. In the present study, the relationship between FRAT1 and VEGFA in angiogenesis was investigated using the human glioblastoma U251 cell line. Small interfering RNAs (siRNAs) were used to silence FRAT1 expression in U251 cells, and the mRNA and protein expression levels of VEGFA, as well as the concentration of VEGFA in U251 cell supernatants, were determined using reverse transcription‑quantitative PCR, western blotting and ELISA. A tube formation assay was conducted to assess angiogenesis. The results demonstrated that siRNA knockdown significantly decreased the protein expression levels of FRAT1 in U251 cells and markedly decreased the mRNA and protein expression levels of VEGFA. Furthermore, the concentration of VEGFA in the cell supernatant was significantly reduced and angiogenesis was suppressed. These results suggested that FRAT1 may promote VEGFA secretion and angiogenesis in human glioblastoma cells via the Wnt/β‑catenin signaling pathway, supporting the potential use of FRAT1 as a promising therapeutic target in human glioblastoma.

STAT1 is a modulator of the expression of frequently rearranged in advanced T-cell lymphomas 1 expression in U251 cells.

Aberrant expression of frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) contributes to poor prognosis in a number of carcinomas. However, its role in glioma remains controversial. In the present study, gene expression profiling was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) functional enrichment and ingenuity pathway analysis (IPA) to evaluate the differential expression of genes and proteins in FRAT1 knockdown U251 glioma cells in comparison with the control. Western blot analysis was conducted to assess the expression levels of FRAT1 and STAT1. A total of 895 downregulated genes were identified in FRAT1-silenced U251 cells. The most enriched processes determined by GO and KEGG analysis of the 895 differentially expressed genes were associated with proliferation, migration and invasion. According to IPA, significant canonical pathways, including the interferon, hepatic fibrosis and Wnt/β-catenin signaling pathways, were identified to be the major enriched pathways. The elevated expression of STAT1 in U251 cells was validated. These results highlighted the regulatory role of FRAT1 in glioma cells with upregulated STAT1 expression.

FRAT1 Enhances the Proliferation and Tumorigenesis of CD133+Nestin+ Glioma Stem Cells In Vitro and In Vivo.

Glioma stem cells (GSCs) are considered the source for development, recurrence, and poor prognosis of glioma, so treatment targeted GSCs is of great interest. The frequently rearranged in advanced T cell lymphomas-1 (FRAT1) gene is an important member of the Wnt/β-catenin signaling transduction pathway, and aberrantly activation of Wnt signaling has been identified to contribute to the tumorigenesis, proliferation, invasion of a variety kinds of cancer stem cells. However, correlations between FRAT1 and GSCs and the specific mechanisms remain unclear. In this study, we aimed to investigate the effect of FRAT1 on GSCs proliferation, colony formation, sphere formation and tumorigenesity in vitro and in vivo and its underlying mechanism. Lentiviral transfection was used to construct GSCs with low FRAT1 expression. The expression of FRAT1 on GSCs proliferation in vitro was assessed by cell counting kit-8(CCK-8). Colony formation and sphere formation assays were conducted to assess the colony and sphere formation ability of GSCs. Then, an intracranial glioma nude mouse model was built to measure the effect of low FRAT1 expression on GSCs proliferation and tumorigenesity in vivo. Real-time PCR, Western blot, and Immunohistochemistry were processed to detect the mRNA and protein expressions of FRAT1, β-catenin in the glioma tissue of xenograft mice to study their correlations. The functional assays verifed that low FRAT1 expression inhibited CD133+Nestin+ GSCs proliferation, colony formation, sphere formation ability in vitro. In vivo GSCs xenograft mice model showed that low FRAT1 expression suppressed the proliferation and tumorigenesity of CD133+Nestin+ GSCs and reduced β-catenin mRNA and protein expression. Furthermore, the expression of FRAT1 and β-catenin were positively correlated. Altogether, results indicate that FRAT1 enhances the proliferation, colony formation, sphere formation and tumorigenesity of CD133+Nestin+ glioma stem cells in vitro and in vivo as well as the expression of β-catenin. Therefore, inhibiting proliferation of GSCs and FRAT1 may be a molecular target to GSCs in treating human glioma in the future.

LncRNA SNHG1 influences cell proliferation, migration, invasion, and apoptosis of non-small cell lung cancer cells via the miR-361-3p/FRAT1 axis.

BackgroundNon‐small‐cell lung cancer (NSCLC) is the most lethal type of cancer. Long non‐coding RNAs (lncRNAs) and microRNAs (miRNAs) have been identified as crucial regulators in the development of NSCLC. The aim of our study was to explore the molecular mechanism of SNHG1 to enable better treatment for NSCLC patients.MethodsQuantitative real‐time polymerase chain reaction (qRT‐PCR) was performed to detect the expression of Small nucleolar RNA host gene 1 (SNHG1), miR‐361‐3p and frequently rearranged in advanced T‐cell lymphomas 1 (FRAT1). The protein level of FRAT1 was measured by western blot assay. Cell proliferation was evaluated by methyl thiazolyl tetrazolium (MTT) assay. Cell apoptosis was assessed by flow cytometry assay. The number of migrated and invaded cells were counted by transwell assay. The relationship between miR‐361‐3p and SNHG1 or FRAT1 was confirmed by dual‐luciferase reporter assay.ResultsOur results indicated that SNHG1 and FRAT1 were highly expressed in NSCLC tissues and cells. SNHG1 silencing inhibited proliferation, induced apoptosis and blocked migration and invasion of NSCLC cells. Also, FRAT1 downregulation suppressed proliferation, promoted apoptosis and hindered migration and invasion of NSCLC cells. Further, FRAT1 could recover the effects of SNHG1 silencing on proliferation, apoptosis, migration and invasion of NSCLC cells. SNHG1 sponged miR‐361‐3p and negatively regulated miR‐361‐3p expression. Meanwhile, miR‐361‐3p targeted FRAT1 and inversely modulated FRAT1 expression. In addition, miR‐361‐3p inhibition abated the effect of SNHG1 knockdown on FRAT1 expression.ConclusionIn conclusion, LncRNA SNHG1 promoted the proliferation, repressed apoptosis and enhanced migration and invasion of NSCLC cells by regulating FRAT1 expression via sponging miR‐361‐3p.

FRAT1 expression regulates proliferation in colon cancer cells.

Colorectal cancer is one of the most common gastric malignancies worldwide. However, the underlying mechanism of colon cancer development and valuable indicators of the disease remain unclear. In this study, the expression of frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) in colon cancer was investigated and the association between FRAT1 expression and biological properties of tumors was analyzed. A total of 147 colon cancer tissue samples and adjacent normal tissues were collected between January 2013 and June 2014. The FRAT1 gene and protein expression levels were analyzed in tissues with different TNM and pathological stages. Small hairpin RNAs (shRNAs) containing the human FRAT1 gene were constructed and transfected into colon cancer HT-29 cells. The proliferation and migration of the cells was also analyzed in relation to a reduction in FRAT1 expression. In colon cancer tissues, the expression of FRAT1 was significantly higher when compared with adjacent tissues. In addition, FRAT1 expression was found to positively correlate with the degree of tumor malignancy, and this difference was determined to be statistically significant (P<0.05). Following shRNA transfection in HT-29 cells to decrease the expression of FRAT1, the proliferation and migration of the HT-29 cells decreased (due to conversion of the shRNA into small interfering RNA). These results indicate that in colon cancer, FRAT1 may present a novel tool for analyzing the tumor progression and may be a novel therapeutic target for the treatment of colon cancer.

Knockdown of FRAT1 inhibits hypoxia-induced epithelial-to-mesenchymal transition via suppression of the Wnt/β-catenin pathway in hepatocellular carcinoma cells.

Hypoxia-induced epithelial-to-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) was investigated. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) is a positive regulator of the Wnt/β-catenin signaling pathway and is overexpressed in many human tumors. However, the expression and role of FRAT1 in HCC has not been elucidated. In this study, we investigated the effect of FRAT1 on EMT process in HCC cells induced by hypoxia. Our results showed that FRAT1 is highly expressed in HCC tissues and cell lines. Hypoxia significantly induced FRAT1 expression in HCC cells. FRAT1 knockdown inhibited hypoxia-induced cell migration/invasion, downregulation of epithelial markers and upregulation of mesenchymal markers. Moreover, FRAT1 knockdown suppressed the expression levels of β-catenin, cyclinD1 and c-myc in HCC cells under the same hypoxic condition. Our results revealed that FRAT1 is a hypoxia factor that is critical for the induction of EMT in HCC cells. These data suggest a potential role for targeting FRAT1 in the prevention of hypoxia-induced HCC cancer progression and metastasis mediated by EMT.

Frequently rearranged in advanced T‑cell lymphomas‑1 demonstrates oncogenic properties in prostate cancer.

Prostate cancer is the fifth most common cause of cancer-associated mortality for males worldwide. Although dysregulation of the β-catenin/T-cell factor (TCF) pathway has been previously reported in prostate cancer, the mechanisms underlying this process remain unknown. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) functions as a positive regulator of the β-catenin/TCF signaling pathway. However, to the best of our knowledge, the molecular association between FRAT1 and the β-catenin/TCF pathway in prostate cancer has not been investigated. In the present study, FRAT1 expression was analyzed in normal prostate tissues and prostate adenocarcinoma samples using publicly available databases, a commercial tissue microarray and immunohistochemistry techniques. In addition, FRAT1 expression levels were altered by overexpression or RNA interference-mediated depletion in prostate cancer cells. The effects of FRAT1 expression on tumor growth were determined using cell growth curves in vitro and xenografts in nude mice in vivo. The effects of FRAT1 on β-catenin/TCF activity were measured using the TOPFLASH reporter assay. FRAT1 was expressed exclusively in the nuclei of normal prostate basal cells, and nuclear FRAT1 was detected in 68% (40/59) of prostate adenocarcinoma samples. In addition, FRAT1 activated the TCF luciferase reporter gene promoter in prostate cancer cells, and was observed to promote the growth of prostate cancer cells in vitro. Furthermore, FRAT1 expression was sufficient to transform NIH3T3 mouse embryonic fibroblast cells and lead to tumor formation in vivo. These results suggest that FRAT1 demonstrates oncogenic properties in prostate cancer, potentially by suppressing the inhibitory effect of nuclear glycogen synthase 3β against β-catenin/TCF activity, thus activating the Wnt/β-catenin signaling pathway and promoting cell growth.

Silencing of FRAT1 by siRNA inhibits the proliferation of SGC7901 human gastric adenocarcinoma cells.

Frequently rearranged in advanced T cell lymphomas-1 (FRAT1) positively regulates the Wnt/β-catenin signaling pathway by inhibiting glycogen synthase kinase-3 mediated phosphorylation of β-catenin. FRAT1 is a proto-oncogene, implicated in tumorigenesis. The present study aimed to investigate the effects of FRAT1 silencing on the proliferation and apoptosis of SGC7901 cells. FRAT1 in SGC7901 cells was silenced by RNA interference. Reverse transcription-quantitative polymerase chain reaction was used for the analysis of FRAT1 mRNA and western blotting was used to evaluate FRAT1 and β-catenin protein levels. Cell proliferation was analyzed by the MTT assay. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The expression of FRAT1 mRNA, FRAT1 and β-catenin protein in FRAT1-silenced SGC7901 cells were reduced significantly compared to untreated cells. The proliferation of FRAT1 silenced SGC7901 cells decreased significantly The FRAT1 silenced SGC7901 cells were arrested at G0/G1 stage to a greater degree, and apoptosis was increased. In summary, silencing of FRAT1 inhibits SGC7901 cell proliferation and induces apoptosis, possible through a reduction in β-catenin expression. FRAT1 may serve as a prognostic biomarker and therapeutic target for gastric cancer.

The clinical significance of FRAT1 and ABCG2 expression in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with intrinsic resistance to cytotoxic agents. The molecular mechanisms associated with high malignancy and resistance to chemotherapy and radiotherapy have not been fully elucidated. This study investigated the clinicopathological significances of frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) and ATP-binding cassette subfamily G member 2 (ABCG2) expression in PDAC. FRAT1 and ABCG2 protein expression in 106 PDAC, 35 peritumoral tissues, 55 benign pancreatic tissues, and 13 normal pancreatic tissues was measured by immunohistochemistry. FRAT1 and ABCG2 protein was overexpressed in PDAC tumors compared to peritumoral tissues, benign pancreatic tissues, and normal pancreatic tissues (P < 0.01). The percentage of cases with positive FRAT1 and ABCG2 overexpression was significantly higher in PDAC patients with poor differentiation, lymph node metastasis, invasion, and TNM stage III/IV disease than in patients with well-differentiated tumor, no lymph node metastasis and invasion, and TNM stage I/II disease (P < 0.05 or P < 0.01). In pancreatic tissues with benign lesions, tissues with positive FRAT1 and ABCG2 protein expression exhibited dysplasia or intraepithelial neoplasia. Kaplan-Meier survival analysis showed that PDAC patients with positive FRAT1 and ABCG2 expression survived significantly shorter than patients with negative FRAT1 and ABCG2 expression (P < 0.05 or P < 0.001). Cox multivariate analysis revealed that positive FRAT1 and ABCG2 expression was an independent poor prognosis factor in PDAC patients. FRAT1 and ABCG2 overexpression is associated with carcinogenesis, progression, and poor prognosis in patients with PDAC.

FRAT1 overexpression leads to aberrant activation of β‐catenin/TCF pathway in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with a poor prognosis. Although aberrant activation of beta-catenin/T-cell factor (TCF) pathway has been observed in ESCC, mechanisms underlying this phenomenon remain unknown. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1), overexpressed in some ESCC lines, is a positive regulator of beta-catenin/TCF pathway. However, little is known about the molecular relationship between FRAT1 and beta-catenin/TCF in ESCC. In this study, we analyzed freshly resected ESCC specimens and demonstrated that FRAT1 was overexpressed in approximately 74% of tumor samples compared with matched normal tissue. Overexpression of FRAT1 significantly promoted esophageal cancer cells growth, whereas suppression of FRAT1 level by RNAi markedly inhibited their growth. In addition, FRAT1 overexpression induced the nuclear accumulation of beta-catenin and promoted the transcriptional activity of beta-catenin/TCF. These effects were reversed by coexpression of GSK 3beta or DeltaN TCF4. Furthermore, accumulation of beta-catenin was correlated with FRAT1 overexpression in ESCC and the basal layer of normal esophageal epithelium. Finally, continued expression of c-Myc is necessary and sufficient for maintenance of the growth state in cells expressing FRAT1. Taken together, these results support the novel hypothesis that aberrant activation of beta-catenin/TCF pathway in esophageal cancer appears to be due to upstream events such as FRAT1 overexpression, and c-Myc may be an important element in oncogenesis of human ESCC induced by FRAT1.

Tissue microarray analysis of human FRAT1 expression and its correlation with the subcellular localisation of β-catenin in ovarian tumours

The mechanisms involved in the pathogenesis of ovarian cancer are poorly understood, but evidence suggests that aberrant activation of Wnt/β-catenin signalling pathway plays a significant role in this malignancy. However, the molecular defects that contribute to the activation of this pathway have not been elucidated. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) is a candidate for the regulation of cytoplasmic β-catenin. In this study, we developed in situ hybridisation probes to evaluate the presence of FRAT1 and used an anti-β-catenin antibody to evaluate by immunohistochemistry the expression levels and subcellular localisation of β-catenin in ovarian cancer tissue microarrays. Expression of FRAT1 was found in some human normal tissues and 47% of ovarian adenocarcinomas. A total of 46% of ovarian serous adenocarcinomas were positive for FRAT1 expression. Accumulation of β-catenin in the nucleus and/or cytoplasm was observed in 55% ovarian adenocarcinomas and in 59% of serous adenocarcinomas. A significant association was observed in ovarian serous adenocarcinomas between FRAT1 and β-catenin expression (P<0.01). These findings support that Wnt/β-catenin signalling may be aberrantly activated through FRAT1 overexpression in ovarian serous adenocarcinomas. The mechanism behind the overexpression of FRAT1 in ovarian serous adenocarcinomas and its significance is yet to be investigated.

FRAT1 peptide decreases Aβ production in swAPP751 cells

Recently, LiCl has been shown to inhibit amyloid β peptide secretion in association with diminished glycogen synthase kinase β (GSK3β) activity. However, it remains unclear if direct inhibition of GSK3β activity will result in decreased Aβ production. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) protein is a negative regulator of GSK3α/β kinase activity. To examine whether direct inhibition of GSK3α/β kinase activity can lower Aβ production, a FRAT1 peptide was expressed in swAPP751 cells that produce high levels of Aβ. Our data demonstrate that cellular expression of FRAT1 peptide in swAPP751 cells increases both GSK3α and β phosphorylation on Ser21 and Ser9, respectively, while inhibiting kinase activity of both isoforms. Moreover, as a result of FRAT1 expression, the production of both total Aβ and Aβ1-42 was significantly decreased. Thus, we provide evidence that direct regulation of GSK3α/β by FRAT1 peptide significantly decreases Aβ production in swAPP751 cells.

A GSK3-binding peptide from FRAT1 selectively inhibits the GSK3-catalysed phosphorylation of Axin and β-catenin

The Axin-dependent phosphorylation of β-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects β-catenin against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) is a mammalian homologue of a GSK3-binding protein (GBP), which appears to play a key role in the correct establishment of the dorsal-ventral axis in Xenopus laevis. Here, we demonstrate that FRATtide (a peptide corresponding to residues 188–226 of FRAT1) binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin and β-catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a ‘priming’ phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of β-catenin phosphorylation.