EBV miRNAs BART11 and BART17-3p promote immune escape through the enhancer-mediated transcription of PD-L1.

Epstein-Barr virus (EBV) is reportedly the first identified human tumor virus, and is closely related to the occurrence and development of nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and several lymphomas. PD-L1 expression is elevated in EBV-positive NPC and GC tissues; however, the specific mechanisms underlying the EBV-dependent promotion of PD-L1 expression to induce immune escape warrant clarification. EBV encodes 44 mature miRNAs. In this study, we find that EBV-miR-BART11 and EBV-miR-BART17-3p upregulate the expression of PD-L1 in EBV-associated NPC and GC. Furthermore, EBV-miR-BART11 targets FOXP1, EBV-miR-BART17-3p targets PBRM1, and FOXP1 and PBRM1 bind to the enhancer region of PD-L1 to inhibit its expression. Therefore, EBV-miR-BART11 and EBV-miR-BART17-3p inhibit FOXP1 and PBRM1, respectively, and enhance the transcription of PD-L1 (CD274, http://www.ncbi.nlm.nih.gov/gene/29126 ), resulting in the promotion of tumor immune escape, which provides insights into potential targets for EBV-related tumor immunotherapy.

Zinc moderates circular RNA CircFOXP1 expression in order to regulate ferroptosis during lung adenocarcinoma.

The present study aimed to gain insight into putative anticancer effect of dietary zinc in rat lung cancer model by targeting ferroptosis. Ferroptosis is an emerging type of programmed cell death, which activates oxidative cell death in an iron and lipid peroxides-dependent manner. Targeting ferroptosis is a novel therapeutic approach for cancer therapy. Circular RNAs (circRNAs), as a form of noncoding RNAs with a specific closed circular sequence are emerging as a new field in cancer research. However, the regulatory mechanisms of circRNAs in ferroptosis during lung cancer development are still elusive. In this work, we elucidate the potential prognostic value and the crucial role of circular RNA circFOXP1 in ferroptosis during lung cancer and modulatory potential of zinc. We found that the expression of circFOXP1 was remarkably up-regulated in clinical tumorous tissues compared with adjacent tissues. The knockdown of circFOXP1 suppressed the cell viability of lung cancer cells. The colony formation counts of lung cancer cells were repressed by the depletion of circFOXP1 as well. Moreover, the treatment of zinc, repressed the cell viability of lung cancer cells and the overexpression of circFOXP1 rescued the phenotype. Meanwhile, the levels of malondialdehyde (MDA), iron, and lipid ROS were enhanced post zinc treatment. Collectively, we concluded that circular RNA circFOXP1 is a potential diagnostic biomarker and contributes to malignant progression by repressing ferroptosis of lung cancer cells. Further, zinc be served as a promising therapeutic approach against lung cancer.

Enhancer recruitment of transcription repressors RUNX1 and TLE3 by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia.

Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ∼30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.

Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Regulatory T-cells (Tregs) impair cancer immunosurveillance by creating an immunosuppressive environment that fosters tumor cell survival. Our previous findings demonstrated that neutrophil extracellular traps (NETs), which are involved both in innate and adaptive immunity, are abundant in livers affected by non-alcoholic steatohepatitis (NASH). However, how NETs interact with Tregs in the development of NASH-associated hepatocellular carcinoma (NASH-HCC) is not known. METHODS: A choline-deficient, high-fat diet+diethylnitrosamine mouse model and the stelic animal model were utilized for NASH-HCC and a western diet mouse model was used for NASH development. Treg depletion was achieved using FoxP3-DTR mice. RNA sequencing was used to explore the mechanism by which NETs could regulate Treg differentiation. Bioenergetic analyses of naïve CD4+ T-cells were assessed by Seahorse. RESULTS: Although the absolute number of CD4+ T-cells is lower in NASH livers, the Treg subpopulation is selectively increased. Depleting Tregs dramatically inhibits HCC initiation and progression in NASH. There is a positive correlation between increased NET and hepatic Treg levels. RNA sequencing data reveals that NETs impact gene expression profiles in naïve CD4+ T-cells, with the most differentially expressed genes being those involved in mitochondrial oxidative phosphorylation. By facilitating mitochondrial respiration, NETs can promote Treg differentiation. Metabolic reprogramming of naïve CD4+ T-cells by NETs requires toll-like receptor 4. Blockade of NETs in vivo using Pad4-/- mice or DNase I treatment reduces the activity of Tregs. CONCLUSIONS: Tregs can suppress immunosurveillance in the premalignant stages of NASH. NETs facilitate the crosstalk between innate and adaptive immunity in NASH by promoting Treg activity through metabolic reprogramming. Therapies targeting NETs and Treg interactions could offer a potential strategy for preventing HCC in patients with NASH. LAY SUMMARY: Regulatory T-cells (Tregs) can promote tumor development by suppressing cancer immunosurveillance, but their role in carcinogenesis during non-alcoholic steatohepatitis (NASH) progression is unknown. Herein, we discovered that selectively increased intrahepatic Tregs can promote an immunosuppressive environment in NASH livers. Neutrophil extracellular traps (NETs) link innate and adaptive immunity by promoting Treg differentiation via metabolic reprogramming of naïve CD4+ T-cells. This mechanism could be targeted to prevent liver cancer in patients with NASH.

Therapeutic strategies targeting FOXO transcription factors.

FOXO proteins are transcription factors that are involved in numerous physiological processes and in various pathological conditions, including cardiovascular disease, cancer, diabetes and chronic neurological diseases. For example, FOXO proteins are context-dependent tumour suppressors that are frequently inactivated in human cancers, and FOXO3 is the second most replicated gene associated with extreme human longevity. Therefore, pharmacological manipulation of FOXO proteins is a promising approach to developing therapeutics for cancer and for healthy ageing. In this Review, we overview the role of FOXO proteins in health and disease and discuss the pharmacological approaches to modulate FOXO function.

Long non-coding RNA FOXD3-AS1 silencing exerts tumor suppressive effects in nasopharyngeal carcinoma by downregulating FOXD3 expression via microRNA-185-3p upregulation.

Emerging evidence indicates that the incidence of nasopharyngeal carcinoma (NPC) remains high in endemic regions despite changing environmental factors, suggesting that genetic traits contribute to its development. Recently, long non-coding RNA-microRNA-messenger RNA (lncRNA-miRNA-mRNA) axis has been reported to be implicated in the pathophysiological processes of malignancies. Moreover, initial bioinformatic analysis revealed a highly expressed lncRNA Forkhead box D3 antisense RNA1 (FOXD3-AS1) for mechanistic network underlying NPC in this present study. Therefore, this study aims to delineate the ability of lncRNA FOXD3-AS1 to influence the NPC progression. The relationship among lncRNA FOXD3-AS1, miR-185-3p, and FOXD3 was identified with bioinformatics prediction, dual-luciferase reporter gene assays, RNA-binding protein immunoprecipitation, and RNA pull-down assays. Furthermore, effects of lncRNA FOXD3-AS1 on malignant phenotypes in vitro, alongside tumor formation in vivo, of transfected NPC stem-like cells were examined with gain- and loss-of-function experiments. Our findings revealed that lncRNA FOXD3-AS1 and FOXD3 exhibited increased expression levels, while miR-185-3p exhibited diminished levels in NPC. The levels of lncRNA FOXD3-AS1 and FOXD3 were further correlated with tumor node metastasis stage and pathological type of patients with NPC. LncRNA FOXD3-AS1 was also confirmed to negatively regulate the miR-185-3p expression, which further targeted the downstream gene FOXD3. In addition, lncRNA FOXD3-AS1 knockdown repressed cell stemness, colony formation, viability, invasion, migration, and in vivo tumor growth, and accelerated cell apoptosis. Moreover, FOXD3 silencing or miR-185-3p overexpression reversed the effects of lncRNA FOXD3-AS1. Our findings provide evidence indicating that lncRNA FOXD3-AS1 could bind to miR-185-3p to upregulate the FOXD3 expression, thereby promoting the development of NPC.

Extracellular vesicles produced by immunomodulatory cells harboring OX40 ligand and 4-1BB ligand enhance antitumor immunity.

Genetically modified tumor cells harboring immunomodulators may be used as therapeutic vaccines to stimulate antitumor immunity. The therapeutic benefit of these tumor vaccines is extensively investigated and mechanisms by which they boost antitumor response may be further explored. Tumor cells are large secretors of extracellular vesicles (EVs). These EVs are able to vehiculate RNA and proteins to target cells, and engineered EVs also vehiculate recombinant proteins. In this study, we explore immunomodulatory properties of EVs derived from antitumor vaccines expressing the TNFSF ligands 4-1BBL and OX40L, modulating immune response mediated by immune cells and eliminating tumors. Our results suggest that the EVs secreted by genetically modified tumor cells harboring TNFSF ligands can induce T cell proliferation, inhibit the transcription factor FoxP3, associated with the maintenance of Treg phenotype, and enhance antitumor activity mediated by immune cells. The immunomodulatory extracellular vesicles have potential to be further engineered for developing new approaches for cancer therapy.

FOX transcription factor family in hepatocellular carcinoma.

The pathogenesis of hepatocellular carcinoma (HCC) is a multistep process, involving the progressive accumulation of molecular alterations and transcriptomic alterations. The Forkhead-box (FOX) transcription factor family is characterized by its unique DNA binding domain (FKH or winged-helix domain). Human FOX family consists of about 17 subfamilies, at least 43 members. Some of them are liver-enriched transcription factors, suggesting that they may play a crucial role in the development or/and functions of the liver. Dysregulation of FOX transcription factors may contribute to the pathogenesis of HCC because they can activate or suppress the expression of various tumor-related molecules, and pinpoint different molecular and cellular events. Here we summarized, analyzed and discussed the status and the functions of the human FOX family of transcription factors in HCC, aiming to help the further development of them as potential therapeutic targets or/and diagnostic/prognostic markers for HCC.

Functionally analyzing the important roles of hepatocyte nuclear factor 3 (FoxA) in tumorigenesis.

Transcriptional factors (TFs) play a central role in governing gene expression under physiological conditions including the processes of embryonic development, metabolic homeostasis and response to extracellular stimuli. Conceivably, the aberrant dysregulations of TFs would dominantly result in various human disorders including tumorigenesis, diabetes and neurodegenerative diseases. Serving as the most evolutionarily reserved TFs, Fox family TFs have been explored to exert distinct biological functions in neoplastic development, by manipulating diverse gene expression. Recently, among the Fox family members, the pilot roles of FoxAs attract more attention due to their functions as both pioneer factor and transcriptional factor in human tumorigenesis, particularly in the sex-dimorphism tumors. Therefore, the pathological roles of FoxAs in tumorigenesis have been well-explored in modulating inflammation, immune response and metabolic homeostasis. In this review, we comprehensively summarize the impressive progression of FoxA functional annotation, clinical relevance, upstream regulators and downstream effectors, as well as valuable animal models, and highlight the potential strategies to target FoxAs for cancer therapies.

Inhibiting Forkhead box K1 induces autophagy to reverse epithelial-mesenchymal transition and metastasis in gastric cancer by regulating Myc-associated zinc finger protein in an acidic microenvironment.

BACKGROUND: Forkhead box K1 (FOXK1) is a transcription factor belonging to the Forkhead box (FOX) family and is closely related to the development of various cancers, but the functional mechanism through which FOXK1 regulates autophagy and epithelial-mesenchymal transition (EMT) in the acidic microenvironment of gastric cancer (GC) remains unclear. RESULTS: Our results indicated that the inhibition of FOXK1 induced autophagy and thus exerted antimetastatic effects in an acidic microenvironment. The dual inhibition of mammalian target of rapamycin (mTOR) and FOXK1 enhanced autophagy and reversed EMT of acidic GC cells. In addition, FOXK1 activated transcription in conjunction with the MAZ promoter. CONCLUSION: Together, our results suggest that FOXK1 can be used as an independent prognostic indicator for GC patients. We also revealed a new strategy involving the cotargeting of FOXK1 and autophagy to reverse the effects of EMT. MAZ is involved in the development and progression of GC as a downstream target of FOXK1. METHODS: Here, the cellular responses to the inhibition of FOXK1 in GC were studied in vivo and in vitro through wound healing assays, transwell assays, Western blotting, laser confocal microscopy and transmission electron microscopy. The molecular mechanisms of FOXK1 and Myc-associated zinc finger protein (MAZ) were studied via chromatin immunoprecipitation sequencing (ChIP-seq), bioinformatics, Western blotting, and quantitative real-time PCR (q-PCR).

LncRNA TRERNA1 promotes malignant progression of NSCLC through targeting FOXL1.

OBJECTIVE: Previous studies have shown the carcinogenic role of long-chain non-coding RNAs (lncRNA) TRERNA1. However, the role of TRERNA1 in non-small cell lung cancer (NSCLC) has not been reported. This research aims to explore the regulatory effect of TRERNA1/FOXL1 axis on the malignant progression of NSCLC. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression levels of TRERNA1 and FOXL1 in 39 pairs of tumor tissues and paracancerous ones collected from NSCLC patients. The potential relation between TRERNA1 expression and clinical indicators of NSCLC patients was analyzed. Meanwhile, expression levels of TRERNA1 and FOXL1 in NSCLC cell lines were also detected by qRT-PCR. In addition, TRERNA1 knockdown model was constructed in H358 and SPC-A1 cells. Cell counting kit-8 (CCK-8), cell colony formation assay, and flow cytometry were applied to analyze the influence of TRERNA1 on NSCLC cell biological functions. Finally, Dual-Luciferase reporter gene assay and cell reverse recovery experiments were performed to figure out the underlying mechanisms of TRERNA1 in regulating NSCLC progression. RESULTS: QRT-PCR results indicated that the expression level of lncRNA TRERNA1 in tumor tissue samples of NSCLC patients was remarkably higher than that in adjacent tissues. Compared with NSCLC patients with low expression of TRERNA1, patients with high TRERNA1 expression had a worse pathological stage and overall survival. Similarly, compared with cells in sh-NC group, the proliferation ability of cells in sh-TRERNA1 group was remarkably attenuated. In addition, cell ratio in the G1 phase increased after knockdown of TRERNA1, suggesting the arrested G1/S cell cycle. Subsequently, FOXL1 was downregulated in NSCLC cell lines and tumor tissues. Meanwhile, FOXL1 level was verified to be negatively correlated with TRERNA1 level. Additionally, the binding between TRERNA1 and FOXL1 was confirmed by Dual-Luciferase reporter gene assay. Cell reverse investigation indicated the involvement of FOXL1 in TRERNA1-regulated malignant progression of NSCLC. CONCLUSIONS: LncRNA TRERNA1 was up-regulated both in NSCLC tissues and cell lines. Its level was associated with pathological stage and poor prognosis in NSCLC. In addition, lncRNA TRERNA1 could promote the malignant progression of NSCLC via modulating FOXL1.

Treatment of experimental autoimmune encephalomyelitis with engineered bi-specific Foxp3+ regulatory CD4+ T cells.

The use of autoantigen-specific regulatory T cells (Tregs) as a cellular therapy for autoimmune diseases is appealing. However, it is challenging to isolate and expand large quantity of Tregs expressing disease-relevant T-cell receptors (TCR). To overcome this problem, we used an approach aiming at redirecting the specificity of polyclonal Tregs through autoreactive TCR gene transfer technology. In this study, we examined whether Tregs engineered through retroviral transduction to express a TCR cross-reactive to two CNS autoantigens, myelin oligodendrocyte glycoprotein (MOG) and neurofilament-medium (NF-M), had a superior protective efficacy compared with Tregs expressing a MOG mono-specific TCR. We observed that engineered Tregs (engTregs) exhibited in vitro regulatory effects related to the antigenic specificity of the introduced TCR, and commensurate in potency with the avidity of the transduced TCR. In experimental autoimmune encephalomyelitis (EAE), adoptively transferred engTregs proliferated, and migrated to the CNS, while retaining FoxP3 expression. EngTregs expressing MOG/NF-M cross-reactive TCR had superior protective properties over engTregs expressing MOG-specific TCR in MOG-induced EAE. Remarkably, MOG/NF-M bi-specific TCR-engTregs also improved recovery from EAE induced by an unrelated CNS autoantigen, proteolipid protein (PLP). This study underlines the benefit of using TCRs cross-reacting towards multiple autoantigens, compared with mono-reactive TCR, for the generation of engTregs affording protection from autoimmune disease in adoptive cell therapy.

Pembrolizumab Interferes with the Differentiation of Human FOXP3+-Induced T Regulatory Cells, but Not with FOXP3 Stability, through Activation of mTOR.

Programmed cell death 1 (PD-1) is critical for T regulatory cells (Tregs) to maintain peripheral tolerance to self-antigens. In the tumor microenvironment, interaction between PD-1 and its ligands supports tumor immune evasion. Pembrolizumab blocks interactions of PD-1 with its ligands, enhancing antitumor and clinical responses. We and others have reported that pembrolizumab does not affect function or phenotype of thymic-derived Tregs; however, little is known about its effect on extrathymic differentiation of peripheral Tregs. In this study, we investigated the effect of pembrolizumab on in vitro-induced Tregs (iTregs). Our work showed that PD-1 blockade interferes with iTreg differentiation and has no potential effect on the stability of FOXP3 after differentiation. Additionally, we found that both nontreated and pembrolizumab-treated iTregs were suppressive. However, pembrolizumab-treated iTregs were relatively less suppressive in higher Treg ratios and failed to produce IL-10 compared with their nontreated counterparts. Different methods including transcriptomic analyses confirmed that the downregulation of FOXP3 was mediated by activating mTOR and STAT1 and inhibiting MAPK pathways, shifting the iTreg polarization in favor of Th1 and Th17 subsets. To confirm the role of mTOR activation, we found that rapamycin diminished the effect of pembrolizumab-mediated downregulation of FOXP3. Ingenuity pathway analysis revealed that pembrolizumab-treated iTregs showed upregulation of genes promoting DNA repair and immune cell trafficking, in addition to downregulation of genes supporting cellular assembly and organization. To our knowledge, this is the first study to show that pembrolizumab interferes with differentiation of human FOXP3+ iTregs and to disclose some of the molecular pathways involved.

FOXP3 Inhibitory Peptide P60 Increases Efficacy of Cytokine-induced Killer Cells Against Renal and Pancreatic Cancer Cells.

BACKGROUND/AIM: Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. MATERIALS AND METHODS: The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA. RESULTS: P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. CONCLUSION: P60 may potentiate CIK cell cytotoxicity against tumor cells.

miR-186-5p Functions as a Tumor Suppressor in Human Osteosarcoma by Targeting FOXK1.

BACKGROUND/AIMS: Aberrantly expressed miRNAs play a vital role in the development of some cancers, such as human osteosarcoma (OS). However, the detailed molecular mechanisms underlying miR-186-5p-involved osteosarcoma are unclear. METHODS: qRT-PCR and western blot analysis were employed to measure the expressions of miR-186-5p and forkhead box k1 (FOXK1). CCK-8 assay evaluated the effect of miR-186-5p and FOXK1 on cell proliferation. Transwell assay confirmed cell migration and invasion. Eventually, the dual-luciferase reporter assay validated 3'-untranslated region (3'-UTR) of FOXK1 as a direct target of miR-186-5p. RESULTS: Down-regulation of miR-186-5p was identified in OS tissues and cell lines, and negatively correlated with distant metastasis, Enneking stage and poor 5-year prognosis as well as the expression of forkhead box k1 (FOXK1) protein. Further assays demonstrated that miR-186-5p overexpression had inhibitory effects on in-vitro cell proliferation, cell cycle, and in-vivo tumor growth. miR-186-5p overexpression also inhibited the epithelial-tomesenchymal transition (EMT), migration and invasion of OS cells. Importantly, miR-186-5p directly targeted FOXK1 3'-UTR and negatively regulated its expression. Silencing of FOXK1 expression enhanced the inhibitory effects of miR-186-5p on OS cell proliferation, migration and invasion. CONCLUSION: These findings highlighted miR-186-5p as a tumor suppressor in the regulation of progression and metastatic potential of OS, and may benefit the development of therapies targeting miR-186-5p in patients with OS.

The FOXC1/FBP1 signaling axis promotes colorectal cancer proliferation by enhancing the Warburg effect.

Aberrant expression of Forkhead box (FOX) transcription factors plays vital roles in carcinogenesis. However, the function of the FOX family member FOXC1 in maintenance of colorectal cancer (CRC) malignancy is unknown. Herein, FOXC1 expression in CRC specimens in The Cancer Genome Atlas (TCGA) cohort was analyzed and validated using immunohistochemistry with a tissue microarray. The effect of FOXC1 expression on proliferation of and glycolysis in CRC cells was assessed by altering its expression in vitro and in vivo. Mechanistic investigation was carried out using cell and molecular biological approaches. Our results showed that FOXC1 expression was higher in CRC specimens than in adjacent benign tissue specimens. Univariate survival analyses of the patients from whom the study specimens were obtained, and validated cohorts indicated that ectopic FOXC1 expression was significantly correlated with shortened survival. Silencing FOXC1 expression in CRC cells inhibited their proliferation and colony formation and decreased their glucose consumption and lactate production. In contrast, FOXC1 overexpression had the opposite effect. Furthermore, increased expression of FOXC1 downregulated that of a key glycolytic enzyme, fructose-1,6-bisphosphatase 1 (FBP1). Mechanistically, FOXC1 bound directly to the promoter regions of the FBP1 gene and negatively regulated its transcriptional activity. Collectively, aberrant FBP1 expression contributed to CRC tumorigenicity, and decreased FBP1 expression coupled with increased FOXC1 expression provided better prognostic information than did FOXC1 expression alone. Therefore, the FOXC1/FBP1 axis induces CRC cell proliferation, reprograms metabolism in CRCs, and constitutes potential prognostic predictors and therapeutic targets for CRC.

Silencing of FOXO6 inhibits the proliferation, invasion, and glycolysis in colorectal cancer cells.

Forkhead box class O6 (FOXO6) is an important member of FOXO family, which has been demonstrated to be implicated in tumor development. However, the role of FOXO6 in colorectal cancer (CRC) is still unclear. The study aimed to investigate the potential roles of FOXO6 in the development of CRC. Our results showed that FOXO6 was overexpressed in CRC tissues and cell lines. FOXO6 knockdown inhibited cell proliferation, as well as repressed the migration and invasion of CRC cells. Additionally, we found that FOXO6 knockdown altered cellular metabolism by inhibiting glycolysis and promoting mitochondrial respiration. Furthermore, FOXO6 knockdown inhibited the activation of PI3K/Akt/mTOR pathway in CRC cells. The results herein indicated that FOXO6 knockdown inhibited cell proliferation, migration, invasion, and glycolysis in CRC cells. PI3K/Akt/mTOR pathway was involved in the effects of FOXO6 on CRC cells. These findings suggested that FOXO6 might be a potential target for the CRC therapy.

FOXC1 silencing inhibits the epithelial­to­mesenchymal transition of glioma cells: Involvement of ß­catenin signaling.

Glioma is a type of malignant brain tumor. Forkhead box C1 (FOXC1) is a conserved transcription factor that is involved in tumorigenesis; however, the function of FOXC1 in glioma remains unclear. The present study aimed to investigate the effects of FOXC1 silencing on the epithelial­to­mesenchymal transition (EMT) of glioma cells. FOXC1­specific small interfering RNAs were employed to downregulate the expression levels of FOXC1 in glioma cells. The proliferation, migration and invasion of glioma cells were assessed by MTT assay, wound healing assay and Transwell assay. Western blot analysis was performed to reveal the effects of FOXC1 on EMT­associated proteins and ß­catenin signaling. The results revealed that, following FOXC1 silencing, the proliferation, migration and invasion of glioma cells were decreased. The expression levels of EMT­associated proteins were also affected. Further examination demonstrated that ß­catenin signaling was involved in the effects of FOXC1 on glioma cells. Previous results suggested that overexpression of ß­catenin reversed the effects of FOXC1 silencing on glioma cells. The present study demonstrated that FOXC1 may regulate the EMT of glioma cells, potentially via ß­catenin signaling. Therefore, FOXC1 may be a potential therapeutic target for the treatment of glioma.

Knockdown of FOXP1 promotes the development of lung adenocarcinoma.

Lung cancer is one of the most common cancers in the world, which accounts for about 27% of all cancer deaths. However, the mechanisms underlying the pathogenesis of lung cancer cells remain largely elusive. In this study, we examined the role of the Forkhead box protein P1 (FOXP1) in lung cancer development. Our Oncomine analysis shows that FOXP1 is downregulated in lung adenocarcinoma compared with normal lung tissue. Knockdown of FOXP1 promotes the growth and invasion of PC9 and A549 cells by regulating genes of chemokine signaling molecules, including CCR1, ADCY5, GNG7, VAV3, and PLCB1. Simultaneous knockdown of CCR1 and FOXP1 attenuated FOXP1 knockdown-induced increase of lung cancer cell growth. Finally, knockdown of FOXP1 in PC9 cells promotes the tumorigenesis via CCR1 signaling in xenograft mouse model. Taken together, our data suggest that FOXP1 plays important roles in preventing lung adenocarcinoma development via suppressing chemokine signaling pathways.

Down-regulation of Treg by interference of enhances the killing effect of CIK on leukemia cell HL-60.

OBJECTIVE: Cytokine-induced killer cells (CIK) is a type of immune cell with antitumor activity induced by a variety of cytokines. Regulatory T cells (Treg) is a T cell subgroup featured as immunosuppressive function. Existing CIK cultivation system may inevitably induce Treg. Forkhead box protein 3 (Foxp3) is an essential transcription factor for Treg function. This study aimed to investigate the effects of CIK on the leukemia cell HL-60. MATERIALS AND METHODS: This work silenced Foxp3 expression on the basis of CIK induction, aiming to investigate its killing effect on HL-60 cells. Peripheral blood mononuclear cells were separated and differentiated to CIK in vitro. CD3+CD56+ and CD4+CD25+Foxp3+ Treg cells were detected by flow cytometry. CIK cells were co-cultured with HL-60 cells under the effector-target ratio at 20:1, 10:1, and 5:1, respectively. The killing activity of CIK on HL-60 cells was determined by CCK-8 assay. RESULTS: The ratio of CD3+, CD3+CD8+, and CD3+CD56+ cells gradually increased during CIK induction. Foxp3 interference significantly reduced Treg cell ratio on the 7th day (p < 0.05). Treg cell ratio was significantly lower in Foxp3 interference group at 1.62% ± 0.07% compared with control (p < 0.05). The killing activity of CIK on HL-60 cells enhanced following the increase of effector-target ratio. Interference of Foxp3 significantly elevated the killing activity of CIK on HL-60 cells with effector-target ratio dependence (p < 0.05). CIK can effectively suppress HL-60 cell growth. Treg significantly inhibited the anti-tumor effect of CIK. CONCLUSIONS: Interference of Foxp3 expression significantly declined Treg level and attenuated its suppression impact on CIK, thus enhancing the killing effect of CIK on HL-60 cells.