Kozai K, etal., Development. 2023 Jan 15;150(2):dev201095. doi: 10.1242/dev.201095. Epub 2023 Jan 17.
Hemochorial placentation involves the differentiation of invasive trophoblast cells, specialized cells that possess the capacity to exit the placenta and invade into the uterus where they restructure the vasculature. Invasive trophoblast cells arise from a well-defined compartment within the placent
a, referred to as the junctional zone in rat and the extravillous trophoblast cell column in human. In this study, we investigated roles for AKT1, a serine/threonine kinase, in placental development using a genome-edited/loss-of-function rat model. Disruption of AKT1 resulted in placental, fetal and postnatal growth restriction. Forkhead box O4 (Foxo4), which encodes a transcription factor and known AKT substrate, was abundantly expressed in the junctional zone and in invasive trophoblast cells of the rat placentation site. Foxo4 gene disruption using genome editing resulted in placentomegaly, including an enlarged junctional zone. AKT1 and FOXO4 regulate the expression of many of the same transcripts expressed by trophoblast cells, but in opposite directions. In summary, we have identified AKT1 and FOXO4 as part of a regulatory network that reciprocally controls critical indices of hemochorial placenta development.
BACKGROUND: Uncontrol cell growth and proliferation is acknowledged to responsible for cancer-related deaths by disorganizing the balance of growth promotion and growth limitation. Aberrant expression of microRNA play essential roles in cancer development, leads to cell proliferation, growth and sur
vival, and promotes the development of various human tumors, including osteosarcoma. Elucidating the molecular mechanism of this abnormality in osteosarcoma carcinogenesis may improve diagnostic and therapeutic strategies for this malignancy. METHODS: The expression of miR-664 in osteosarcoma cell lines and osteosarcoma tissues was examined using real-time PCR. The effects of miR-664 on osteosarcoma cell proliferation were evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, colony formation and Anchorage-independent growth ability assay. The effect of miR-664 on FOXO4 was determine by luciferase assays and western blot assay. RESULTS: The expression of miR-664 was markedly upregulated in osteosarcoma cell lines and tissues, and upregulation of miR-664 enhanced, whereas downregulation of miR-664 inhibited the proliferation of osteosarcoma cells in vivo. Furthermore, using bioinformatics and biological approaches, we showed that miR-664 directly targeted and suppressed the expression of tumor suppressors FOXO4. CONCLUSIONS: Our findings suggest that miR-664 functions as an oncogene miRNA and has an important role in promoting human osteosarcoma cell proliferation by suppressing FOXO4 expression. These data suggests that miR-664 may represent a novel therapeutic target of microRNA-mediated suppression of cell proliferation in osteosarcoma.
Ho SR, etal., FEBS Lett. 2010 Jan 4;584(1):49-54. doi: 10.1016/j.febslet.2009.11.059. Epub .
The FOXO4 transcription factor plays an important role in cell survival in response to oxidative stress. The regulation of FOXO4 is orchestrated by post-translational modifications including phosphorylation, acetylation, and
ubiquitination. Here, we demonstrate that O-GlcNAcylation also contributes to the FOXO4-dependent oxidative stress response. We show that hydrogen peroxide treatment of HEK293 cells increases FOXO4 association with OGT, the enzyme that adds O-GlcNAc to proteins, causing FOXO4 O-GlcNAcylation and enhanced transcriptional activity under acute oxidative stress. O-GlcNAcylation is known to be protective for cells under stress conditions, including oxidative stress. Our data provide a mechanism of FOXO4 anti-oxidative protection through O-GlcNAcylation.
Li J, etal., BMC Mol Biol. 2015 Dec 29;16:24. doi: 10.1186/s12867-015-0052-6.
BACKGROUND: Dysregulation of microRNA-150 (miR-150) is commonly observed in solid tumor and has been reported to be involved in multiple important biological processes, such as cell proliferation, apoptosis, and metastasis. Elevated miR-150 level was also detected in cervical carcinoma, whereas its
function in cancer progression has not been studied yet. METHODS: The expression of miRNA-150 in cervical carcinoma was compared with normal cervical tissue and using qRT-PCR. The effects of miR-150 on cell cycle and apoptosis, as well as the expression of cycle- and apoptosis-related genes, were determined using flow cytometry, TUNEL assay, qRT-PCR, and Western blot, respectively. The direct target of miR-150 was confirmed using 3' untranslated region (UTR) luciferase reporter assay. RESULTS: miR-150 promotes cervical cancer cell survival and growth, while the inhibition of miR-150 suppresses these actions. miR-150 also induced the cell cycle progression from G1/G0 to S phase, resulting in an enhancement of growth. Several cell cycle- and apoptosis-related genes, CyclinD1, p27, BIM, and FASL were modulated by miR-150. Moreover, miR-150 directly reduced the expression of FOXO4, which regulates the expression of CyclinD1, p27, BIM, and FASL, by targeting its 3' UTR. CONCLUSION: Taken together, our data demonstrated that elevated miR-150 targets FOXO4 expression and therefore regulates multiple genes expression, resulting in cervical cancer cell growth and survival.
Smooth muscle cells (SMCs) modulate their phenotype between proliferative and differentiated states in response to physiological and pathological cues. Insulin-like growth factor-I stimulates differentiation of SMCs by activating phosphoinositide-3-kinase (PI3K)-Akt signaling. Foxo forkhead transcri
ption factors act as downstream targets of Akt and are inactivated through phosphorylation by Akt. We show that Foxo4 represses SMC differentiation by interacting with and inhibiting the activity of myocardin, a transcriptional coactivator of smooth muscle genes. PI3K/Akt signaling promotes SMC differentiation, at least in part, by stimulating nuclear export of Foxo4, thereby releasing myocardin from its inhibitory influence. Accordingly, reduction of Foxo4 expression in SMCs by siRNA enhances myocardin activity and SMC differentiation. We conclude that signal-dependent interaction of Foxo4 with myocardin couples extracellular signals with the transcriptional program for SMC differentiation.
BACKGROUND & AIMS: The sarcomatoid change in cholangiocarcinoma (CC) contributes to more aggressive intrahepatic spread and widespread metastasis. Therefore, the aim of this study was to identify the molecular mechanisms of CC metastasis during tumor progression and sarcomatoid change. METHODS: Usin
g the subtraction suppression hybridization (SSH) method, we identified altered expression of the candidate gene ANXA8 and epidermal growth factor receptor (EGFR) in sarcomatoid CC cells. We assessed ANXA8 expression during the progression of CC in cells and tissues and examined its functional significance by performing in vitro cell experiments and using in vivo animal models. RESULTS: ANXA8 is highly expressed in human and hamster CCs but is down-regulated with tumor dedifferentiation. ANXA8 is transcriptionally down-regulated by epidermal growth factor (EGF), which is correlated with the morphologic changes of the epithelial-to-mesenchymal transition (EMT) in the CC cells. Furthermore, ectopic ANXA8 reverses the morphology of cells, and this is associated with focal adhesion kinase expression and altered F-actin dynamics. EGFR and its downstream targets, phosphatidylinositol-3-kinase and Akt, are linked to the phosphorylation of FOXO4, which leads to the inhibition of ANXA8 transcription. In addition, an in vitro cell invasion assay and in vivo spontaneous metastasis assay reveal that ANXA8 inhibits the cell migratory and metastatic characteristics of CC cells. CONCLUSIONS: These findings suggest that FOXO4 and ANXA8 play key roles in growth factor-mediated tumor progression and metastasis during the EMT change in CC.
Oteiza A and Mechti N, J Interferon Cytokine Res. 2015 Nov;35(11):859-74. doi: 10.1089/jir.2015.0020. Epub 2015 Aug 3.
Activation of innate immune response, induced after the recognition of double-stranded RNA (dsRNA), formed during replication of most viruses, results in intracellular signaling cascades ultimately culminating in the expression of type I interferon (IFN). In this study, we provide the first evidence
that FoxO4 triggers the activation of the innate immune signaling pathway in coupling stimulation of TLR3 and RIG-like receptors by the synthetic dsRNA analog, poly(I:C), to IFN-beta and IFN-induced gene induction, whereas knockdown of FoxO4 had opposite effects. Similar effects of FoxO4 were observed during paramyxovirus-mediated IFN-beta transcriptional induction. We further found that knockdown of FoxO4 did not affect IRF3 and NF-kappaB activation by poly(I:C), suggesting that FoxO4 would act downstream in the signaling pathway. In addition, we show that the IFN-induced TRIM22 ubiquitin ligase targets FoxO4 and antagonizes its activity through an unrelated ubiquitin/autophagosomic-lysosomal pathway. Unexpectedly, TRIM22 knockdown strongly sensitizes cells to dsRNA-induced caspase-dependent apoptosis, as early as 2 h after poly(I:C) stimulation, concomitantly to the inhibition of the expression of the antiapoptotic protein, Bcl-2, indicating that TRIM22 might be a key factor for controlling the cell survival after TLR3 stimulation. Taken together, our data demonstrate that the regulation of FoxO4 protein expression and cell survival by TRIM22 controls TLR3-mediated IFN type I gene induction, preventing excessive antiviral response through dsRNA-induced apoptosis.
Li H, etal., Mol Cell Biol. 2007 Apr;27(7):2676-86. Epub 2007 Jan 22.
Phenotypic modulation of vascular smooth muscle cells (SMCs) in the blood vessel wall from a differentiated to a proliferative state during vascular injury and inflammation plays an important role in restenosis and atherosclerosis. Matrix metalloproteinase 9 (MMP9) is a member of the MMP family of
proteases, which participate in extracellular matrix degradation and turnover. MMP9 is upregulated and required for SMC migration during the development of restenotic and atherosclerotic lesions. In this study, we show that FoxO4 activates transcription of the MMP9 gene in response to tumor necrosis factor alpha (TNF-alpha) signaling. Inhibition of FoxO4 expression by small interfering RNA or gene knockout reduces the abilities of SMCs to migrate in vitro and inhibit neointimal formation and MMP9 expression in vivo. We further show that both the N-terminal, Sp1-interactive domain and the C-terminal transactivation domain of FoxO4 are required for FoxO4-activated MMP9 transcription. TNF-alpha signaling upregulates nuclear FoxO4. Our studies place FoxO4 in the center of a transcriptional regulatory network that links gene transcription required for SMC remodeling to upstream cytokine signals and implicate FoxO4 as a potential therapeutic target for combating proliferative arterial diseases.
Xu MM, etal., Asian Pac J Cancer Prev. 2014;15(9):4013-8.
Because of its importance in tumor invasion and metastasis, the epithelial-mesenchymal transition (EMT) has become a research focus in the field of cancer. Recently, evidence has been presented that FoxO4 might be involved in EMT. Our study aimed to detect the expression of FoxO4, E-cadherin and v
imentin in non-small cell lung cancers (NSCLCs). We also investigated clinical features and their correlations with the markers. In our study, FoxO4, E-cadherin and vimentin were assessed by immunohistochemistry in a tissue microarray (TMA) containing 150 cases of NSCLC. In addition, the expression level of FoxO4 protein was determined by Western blotting. The percentages of FoxO4, E-cadherin and vimentin positive expression in NSCLCs were 42.7%, 38.7% and 55.3%, respectively. Immunoreactivity of FoxO4 was low in NSCLC when compared with paired normal lung tissues. There were significant correlations between FoxO4 and TNM stage (P<0.001), histological differentiation (P=0.004) and lymph node metastasis (P<0.001), but no significant links with age (P=0.323), gender (P=0.410), tumor size (P=0.084), smoking status (P=0.721) and histological type (P=0.281). Our study showed that low expression of FoxO4 correlated with decreased expression of E-cadherin and elevated expression of vimentin. Cox regression analysis indicated FoxO4 to be an independent prognostic factor in NSCLC (P=0.046). These data suggested that FoxO4 might inhibit the process of EMT in NSCLC, and might therefore be a target for therapy.
Zhang T, etal., Pathol Res Pract. 2016 Mar;212(3):153-61. doi: 10.1016/j.prp.2015.12.012. Epub 2015 Dec 23.
The capability for DNA double-strand breaks (DSBs) repair is crucial for chromatin dramatic changes and DNA damage in normal and tumor cells. We have investigated the clinicopathological significance of DNA repair gene Ku70 expression in hepatocellular carcinoma. We demonstrated that Ku70 expression
was significantly increased in HCC, and the high expression levels were significantly correlated with gender, maximal tumor size, HBsAg status, tumor nodule number, distant metastasis and Ki-67 expression by clinicopathological analysis. The Kaplan-Meier survival curves revealed that increasing Ku70 expression was associated with poor prognosis in HCC patients. Ku70 expression was an independent prognostic marker of overall HCC patient survival in a multivariate analysis. In addition, through serum starvation and refeeding, we found that Ku70 was lowly expressed in serum-starved Huh7 and HepG2 HCC cells, and was progressively increased after serum-additioning. Furthermore, knockdown of Ku70 inhibited cell proliferation accompanying an increase in p27(kip1) levels through interacting with FOXO4. These findings provide a rational framework for the progression of HCC and could be a potential molecular therapy by targeting the Ku70-FOXO4 interaction.
Chang B, etal., Int J Mol Med. 2013 Mar;31(3):569-76. doi: 10.3892/ijmm.2013.1229. Epub 2013 Jan 4.
Forkhead box 'Other' (FoxO) proteins, a subgroup of the Forkhead transcription factor family, play an important role in mediating the effects of insulin and growth factors on diverse physiological functions. In this study, we investigated the role of FoxO4 in the relationship between alcohol liver d
isease and intestinal barrier dysfunction using an animal model. Six to eight-week-old male WT rats were divided into eight groups. They were separately administered corn starch dissolved in PBS; 40% alcohol (5 g/kg body weight) through stomach feeding every 12 h/time, three times in total; tumor necrosis factor alpha (TNFalpha) (10 microg/kg) injected intraperitoneally 30 min before alcohol administration; wortmannin (1.4 mg/kg) 30 min before alcohol administration; IGF-1 (0.2 mg/kg) 30 min before alcohol administration; anti-TNFalpha (5 mg/kg) injected intravenously 30 min before alcohol administration. In addition, two placebo groups were treated with PBS either intraperitoneally or intravenously prior to alcohol administration. TNFalpha and endotoxin in plasma were measured by ELISA and Tachypleus Amebocye Lysate assays. Immunohistochemistry and western blotting were used to identify the mechanisms of FoxO4 action in regulating epithelial permeability. Furthermore, electron microscopy, reverse transcription-polymerase chain reaction and western blotting were used to examine the expression of tight junction proteins and nuclear factor-kappaB (NF-kappaB). Compared with the control group, TNFalpha in the alcohol group was significantly higher. TNFalpha could induce FoxO4 phosphorylation; p-FoxO4 was limited into the cytoplasm and inactivated; inactive FoxO4 which was in high levels lost the ability to suppress NF-kappaB. Therefore, the expression of NF-kappaB was increased and it downregulated tight junction protein (including ZO-1 and occludin) expression, and increased epithelial permeability. As a result, intestinal bacteria grew excessively, endotoxin was released into the portal circulation and liver injury deteriorated. These results indicate that a complex network of mechanisms is involved in the beneficial effects of FoxO4 in epithelial barrier dysfunction. TNFalpha can upregulate phosphorylation of FoxO4. FoxO4 which is located in the nucleus is limited into the cytoplasm and inactivated; it loses the ability to suppress NF-kappaB activity, it downregulates the expression of tight junction proteins and increases epithelial permeability. Disruption of the intestinal barrier allows endotoxin and other bacterial products in the gut lumen to pass into the portal circulation and cause hepatic inflammation. At the same time, the changes of liver injury deteriorate.
