FOXO3a Transcription Research Articles

Skeletal muscle fibre type-dependent effects of atorvastatin on the PI3K/Akt/mTOR signalling pathway and atrophy-related genes in rats

BackgroundOne of the probable causes of statin myotoxicity is an imbalance between protein synthesis and degradation. These processes are regulated by the PI3K/Akt/mTOR pathway and the ubiquitin‒proteasome system (UPS). The aim of this study was to assess whether the effects of atorvastatin on PI3K/Akt/mTOR pathway downstream proteins, the FoxO3a transcription factor and the UPS genes, i.e., MuRF-1 and MAFbx, depend on muscle fibre type.Methods and resultsAtorvastatin (50 mg/kg) was administered to Wistar rats. The levels of selected PI3K/Akt/mTOR pathway proteins were assayed via Western blotting, whereas MuRF-1, MAFbx and FoxO3a mRNA levels were measured using reverse transcription quantitative polymerase chain reaction (RT‒qPCR). Gomöri trichrome staining was performed to assess skeletal muscle pathology. A decrease in the P-Akt/Akt ratio was observed in the gastrocnemius muscle (MG), whereas an increase in the P-Akt/Akt ratio was observed in the soleus muscle (SOL). FoxO3a gene expression increased in the SOL and extensor digitorum longus (EDL) muscles. MuRF-1 gene expression increased in the MG, and MAFbx expression increased in the EDL. No histopathological changes were observed in any of the tested muscles.ConclusionsIn the absence of overt muscle damage, atorvastatin decreased the P-Akt/Akt ratio in the MG, indicating an increase in inactive Akt. Consistent with the decrease in Akt activation, rpS6 phosphorylation decreased. In SOL, atorvastatin increased the P-Akt/Akt ratio, indicating Akt activation. P-FoxO3a and the P-FoxO3a/FoxO3a ratio increased, suggesting that FoxO3a inactivation occurred. Moreover, in the SOL, atorvastatin did not affect the expression of atrophy-related genes. These findings indicate that atorvastatin has no adverse effect on the Akt pathway in the SOL. Our results showed that the effects of atorvastatin on the Akt signalling pathway and atrophy-related gene expression depend on muscle type.

Anti-Müllerian hormone induces autophagy to preserve the primordial follicle pool in mice.

The reserve pool of primordial follicles (PMFs) is finely regulated by molecules implicated in follicular growth or PMF survival. Anti-Müllerian hormone (AMH), produced by granulosa cells of growing follicles, is known for its inhibitory role in the initiation of PMF growth. We observed in a recent invivo study that injection of AMH into mice seemed to induce an activation of autophagy. Furthermore, injection of AMH into mice activates the transcription factor FOXO3A which is also known for its implication in autophagy regulation. Many studies highlighted the key role of autophagy in the ovary at different stages of folliculogenesis, particularly in PMF survival. Through an invitro approach with organotypic cultures of prepubertal mouse ovaries, treated or not with AMH, we aimed to understand the link among AMH, autophagy, and FOXO3A transcription factor. Autophagy and FOXO3A phosphorylation were analyzed by western blot. The expression of genes involved in autophagy was quantified by RT-qPCR. In our invitro model, we confirmed the decrease in FOXO3A phosphorylation and the induction of autophagy in ovaries incubated with AMH. AMH also induces the expression of genes involved in autophagy. Interestingly, most of these genes are known to be FOXO3A target genes. In conclusion, we have identified a new role for AMH, namely the induction of autophagy, probably through FOXO3A activation. Thus, AMH protects the ovarian reserve not only by inhibiting the growth of PMFs but also by enabling their survival through activation of autophagy.

Role of FOXO3a Transcription Factor in the Regulation of Liver Oxidative Injury.

Oxidative stress has been identified as a key mechanism in liver damage caused by various chemicals. The transcription factor FOXO3a has emerged as a critical regulator of redox imbalance. Multiple post-translational changes and epigenetic processes closely regulate the activity of FOXO3a, resulting in synergistic or competing impacts on its subcellular localization, stability, protein-protein interactions, DNA binding affinity, and transcriptional programs. Depending on the chemical nature and subcellular context, the oxidative-stress-mediated activation of FOXO3a can induce multiple transcriptional programs that play crucial roles in oxidative injury to the liver by chemicals. Here, we mainly review the role of FOXO3a in coordinating programs of genes that are essential for cellular homeostasis, with an emphasis on exploring the regulatory mechanisms and potential application of FOXO3a as a therapeutic target to prevent and treat liver oxidative injury.

Targeted ablation of Fn14 receptor improves exercise capacity and inhibits neurogenic muscle atrophy.

Skeletal muscle atrophy is a prevalent complication in multiple chronic diseases and disuse conditions. Fibroblast growth factor-inducible 14 (Fn14) is a member of the TNF receptor superfamily and a bona fide receptor of the TWEAK cytokine. Accumulating evidence suggests that Fn14 levels are increased in catabolic conditions as well as during exercise. However, the role of Fn14 in the regulation of skeletal muscle mass and function remains poorly understood. In this study, through the generation of novel skeletal muscle-specific Fn14-knockout mice, we have investigated the muscle role of Fn14 in the regulation of exercise capacity and denervation-induced muscle atrophy. Our results demonstrate that there was no difference in skeletal muscle mass between control and muscle-specific Fn14-knockout mice. Nevertheless, the deletion of Fn14 in skeletal muscle significantly improved exercise capacity and resistance to fatigue. This effect of Fn14 deletion is associated with an increased proportion of oxidative myofibers and higher capillaries number per myofiber in skeletal muscle. Furthermore, our results demonstrate that targeted deletion of Fn14 inhibits denervation-induced muscle atrophy in adult mice. Deletion of Fn14 reduced the expression of components of the ubiquitin-proteasome system and non-canonical NF-kappa B signaling in denervated skeletal muscle, as well as increased the phosphorylation of Akt kinase and FoxO3a transcription factor. Collectively, our results demonstrate that targeted inhibition of Fn14 improves exercise tolerance and inhibits denervation-induced muscle atrophy in adult mice.

Inhibition of the cardiac fibroblast-enriched histone methyltransferase Dot1L prevents cardiac fibrosis and cardiac dysfunction

BackgroundCardiac fibrosis is characterized by excessive extracellular matrix deposition that contributes to compromised cardiac function and potentially heart failure. Disruptor of telomeric silencing 1-like (Dot1L) is the catalytic enzyme required for histone H3K79 methylation which has been demonstrated to play a role in transcriptional activation. However, the functions of Dot1L in the process of cardiac fibrosis still remain unknown.ResultsIn the present study, we found that endogenous Dot1L is upregulated in cardiac fibroblasts (CFs) treated with angiotensin II (Ang II) or transforming growth factor (TGF)-β1, along with elevated extracellular matrix (ECM) such as fibronectin, collagen I and III. Silencing or inhibiting Dot1L mitigated Ang II-induced myofibroblast generation and fibrogenesis. We identified the transcription factor-forkhead box O (FoxO) 3a as a novel substrate of Dot1L, the transcriptional activating mark H3K79me3 level on the promoter of FoxO3a was increase in activated-CFs, and inhibition of Dot1L markedly decreased FoxO3a transcription accompanied by a significant decrease in the expression of fibrogenic gene. Knockdown of FoxO3a could alleviate ECM deposition induced by Ang II, on the contrary, overexpression FoxO3a resulting in CFs activation. Consistently, in vivo Dot1L ablation rescued myocardial ischemia-induced cardiac fibrosis and improved cardiac function.ConclusionsOur findings conclude that upregulation of Dot1L results in activation of the cardiac fibroblasts to promote profibrotic gene, eventually causes cardiac fibrosis. Pharmacological targeting for Dot1L might represent a promising therapeutic approach for the treatment of human cardiac fibrosis and other fibrotic diseases.

Protein Hydrolysate from Spirulina platensis Prevents Dexamethasone-Induced Muscle Atrophy via Akt/Foxo3 Signaling in C2C12 Myotubes.

Loss of muscle mass is the primary symptom of sarcopenia. Protein intake is recommended to prevent muscle mass loss, and Spirulina platensis, a microalga with high protein content, is a potential protein supplement. Here, we evaluated the differentiation ability of C2C12 cells and the inhibitory effect of Spirulina hydrolysates (SPH) prepared by Collupulin on dexamethasone (DEX)-treated C2C12 cells. SPH contained 578.27 mg/g protein and 92.30 mg/g branched-chain amino acids. SPH increased C2C12 myotube length and diameter, likely owing to increased MyoD1 and Myf5 expression. Inhibition of increased Atrogin-1, MuRF-1, and FoxO3 expression by SPH in DEX-treated C2C12 cells suppressed DEX-induced muscle atrophy. Moreover, SPH inhibited the DEX-induced increase in cytosolic p-Akt protein expression and suppressed the increase in nuclear FoxO3a protein expression, thereby suppressing the increase in the protein expression of the ubiquitin-proteasome-related factors Atrogin-1 and MuRF-1, which are involved in muscle atrophy. SPH suppressed DEX-induced muscle atrophy by activating the Akt/FoxO3a pathway. SPH promoted C2C12 myoblast differentiation into myotubes and inhibited DEX-induced myotube atrophy by suppressing Atrogin-1 and MuRF-1 expression and regulating the FoxO3a transcription factor. Collectively, SPH can be used as a functional food to inhibit muscle atrophy and promote muscle regeneration.

Modulatory Effects of Alpha-Mangostin Mediated by SIRT1/3-FOXO3a Pathway in Oxidative Stress-Induced Neuronal Cells.

Backgroundalpha-Mangostin, a polyphenolic xanthone, is primarily found in the pericarp of mangosteen throughout Southeast Asia and is considered as the “Queen of Fruit” in Thailand. Nonetheless, it is not clarified how alpha-mangostin protects neuronal cells against oxidative stress.ObjectiveIn this study, molecular mechanisms underlying the neuroprotective effect of alpha-mangostin in defending hydrogen peroxide (H2O2)-induced neurotoxicity was explored.Methodscytotoxicity, reactive oxygen species (ROS) generation, apoptotic cascades, and protein expression profiles were performed incorporation of molecular docking.ResultsHuman SH-SY5Y cells were pretreated with 1 μM alpha-mangostin for 3 h prior to exposure to 400 μM H2O2. alpha-Mangostin significantly inhibited oxidative stress-induced cell death in neuronal cells by reducing BAX protein, decreasing caspase-3/7 activation, and increasing anti-apoptotic BCL-2 protein. Collectively, alpha-mangostin was demonstrated to be a prominent ROS suppressor which reversed the reduction of antioxidant enzymes (CAT and SOD2). Surprisingly, alpha-mangostin significantly promoted the expression of the sirtuin family and the FOXO3a transcription factor exerting beneficial effects on cell survival and longevity. A molecular docking study predicted that alpha-mangostin is directly bound to the active site of SIRT1.ConclusionFindings from this study suggest that alpha-mangostin potentially serves as a promising therapeutic compound against oxidative stress by activation of the SIRT1/3-FOXO3a pathway comparable to the effect of memantine, an anti-AD drug used for the treatment of moderate to severe dementia.

FoxO3a Inhibits Tamoxifen-Resistant Breast Cancer Progression by Inducing Integrin α5 Expression.

Simple SummaryTamoxifen is a mainstay of adjuvant treatment for estrogen receptor α-positive (ERα+) breast tumors, which account for over 70% of all the diagnosed breast malignancies. Unfortunately, ~30% of patients initially responding to the therapy will eventually relapse with an endocrine-resistant disease. Here, we show that the transcription factor FoxO3a can overcome tamoxifen resistance by inhibiting cell motility and invasiveness, the main features of tumor progression. The underlying mechanism could be ascribed to FoxO3a-dependent transcriptional up-regulation of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion. Indeed, FoxO3a and integrin α5 expression is significantly correlated in ERα+ tumors, and FoxO3a protective effects are lost in cells with reduced levels of integrin α5. Therefore, a pharmacological increase in FoxO3a levels could represent an effective approach to be exploited in combination with tamoxifen treatment in order to restore the sensitivity to the therapy in refractory tumors.Resistance to endocrine therapy is still a major clinical challenge in the management of estrogen receptor α-positive (ERα+) breast cancer (BC). Here, the role of the Forkhead box class O (FoxO)3a transcription factor in tumor progression has been evaluated in tamoxifen-resistant BC cells (TamR), expressing lower levels of FoxO3a compared to sensitive ones. FoxO3a re-expression reduces TamR motility (wound-healing and transmigration assays) and invasiveness (matrigel transwell invasion assays) through the mRNA (qRT-PCR) and protein (Western blot) induction of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion and signaling. The induction occurs through FoxO3a binding to a specific Forkhead responsive core sequence located on the integrin α5 promoter (cloning, luciferase, and ChIP assays). Moreover, FoxO3a failed to inhibit migration and invasion in integrin α5 silenced (siRNA) cells, demonstrating integrin α5 involvement in both processes. Finally, using large-scale gene expression data sets, a strong positive correlation between FoxO3a and integrin α5 in ERα+, but not in ER-negative (ERα−), BC patients emerged. Altogether, our data show how the oncosuppressor FoxO3a, by increasing the expression of its novel transcriptional target integrin α5, reverts the phenotype of endocrine-resistant BC toward a lower aggressiveness.

SGK1 Inhibition Induces Fetal Hemoglobin in Erythroid Cells

Introduction: Novel and safe therapeutic targets to increase expression of fetal hemoglobin (HbF) have potential to treat b-hemoglobinopathies (Platt, Brambilla et al. 1994, Steinberg 2020), including sickle cell disease (SCD) in which red blood cell (RBC) hemoglobin S resulting from a mutation in the hemoglobin β-globin subunit causes RBC sickling and hemolysis triggering vascular inflammation (Piel, Steinberg et al. 2017, Kato, Piel et al. 2018). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase in the AGK kinase family that controls physiological processes such as cell growth, proliferation, migration, and apoptosis (Hayashi, Tapping et al. 2001, Sang, Kong et al. 2020). SGK1 is regulated by multiple ligands (insulin, cAMP, IGF-1, steroids, IL-2 and TGF-β) and phosphorylation by SGK1 modulates the activity of downstream effectors including ion channels (ENaC), Na-Cl cotransporters (NCC), membrane transporters, cellular enzymes (GSK3B) and transcription factors (FOXO3a, β-catenin, NF-κB and SP1) (Brunet, Park et al. 2001, Snyder, Olson et al. 2002, Loffing, Flores et al. 2006, Bruhn, Pearson et al. 2010, Boccitto and Kalb 2011, Wang, Hu et al. 2017). Previous studies show that SGK1 mediates survival signals in HEK cells by inhibiting FOXO3a through phosphorylation at Ser-315 (Brunet, Park et al. 2001). Recently, metformin was shown to induce HbF in erythroid cells through FOXO3a activation and metformin prevents RBC sickling in SCD (Zhang, Paikari et al. 2018). Thus, we hypothesized that inhibition of SGK1 and subsequent alleviation of SGK1-induced FOXO3a inhibition, may induce expression of erythroid cell HbF.Methods: We studied the ability of SGK1 to inhibit HbF induction in erythroid cells by culturing CD34+ hematopoietic progenitor stem cells from both healthy and SCD blood donors using a 21-day differentiation protocol. After confirming expression of SGK1 in CD34+ cells by Western blot, SGK1 activity was inhibited using the selective and potent SGK1 inhibitor RA04075215A (Halland, Schmidt et al. 2015). SGK1 is activated by phosphorylation at Thr256 and we confirmed target engagement through measurement of Thr256 phosphorylation on Western blots. To decipher the effect of SGK1 inhibition on the SGK1 downstream pathway, we assessed the inhibition of FOXO3a triggered by SGK1 through evaluation of FOXO3a phosphorylation Ser315. In parallel, we quantified HbF gene transcripts by qPCR, determined the level of HbF protein by Western blot, and quantified F-cells by flow cytometry. Finally, to evaluate the effect of SGK1 inhibition on RBC sickling, we performed a cell sickling assay upon completion of erythroid differentiation in culture. Fully differentiated CD34+ cells from SCD blood donors were incubated under in hypoxia (2% O 2) for 4 hours and then abnormal shaped cells were analyzed using the Amnis® ImageStream® flow cytometer.Results: By day 21 of differentiation, HbF protein expression in CD34+ cells increases significantly in RA04075215A-treated cells versus untreated controls. In addition, a combination of SGK1 inhibition and hydroxyurea treatment reveals a potential synergistic induction of HbF. Western blot analysis shows a decrease in phospho-SGK1 phosphorylated at Thr-256 with SGK1 inhibition, confirming target engagement and loss of SGK1 activity. Downstream of SGK1, phospho-FOXO3a phosphorylated at Ser-315 was also decreased significantly following SGK1 inhibition, demonstrating alleviation of FOXO3a inhibition. Finally, in the RBC sickling assay, RA04075215A-treated cells were significantly protected from sickling under hypoxia compared to controls.Conclusion: In summary, this study establishes SGK1 as a potential new therapeutic target in SCD. We demonstrate that SGK1 inhibition induces HbF in CD34+ cells through FOXO3a transcription factor activation and prevents CD34+ cells from sickling. In the future, in vivo studies are necessary to confirm the role of SGK1 in HbF induction and to assess the efficacy of SGK1 inhibition in improving markers of SCD. DisclosuresNo relevant conflicts of interest to declare.

Effects of the FOXO3a rs 4946936 Gene Polymorphism on the FOXO3a Transcription Factor in Chronic Granulocytic Leukemia Patients

Introduction : FOXO3a has an important role in the maintenance of leukemic stem cells. BCR-ABL inhibition therapy by TKI dasatinib aims to reduce the phosphorylation of the transcription factor FOXO3a, promote localization of FOXO3a in the nucleus and restore transcriptional activity. However, some studies showed that the TKI dasatinib, in addition to increase the relocation of Foxo3a to the intranuclear, also increase the expression of CDKN1c/p57 and Bcl6 genes that became the down target for Foxo3a intra nucleus ATM. Therefore, current therapy is mostly directed at personalized therapy (personalized medicine). The aim of this study was to investigate the effect of the FOXO3a rs4946936 gene polymorphism on the Foxo3a transcription factor in chronic granulocytic leukemia patients treated with Imatinib mesylate.
 Method : This is a cross-sectional study in patients with chronic granulocytic leukemia (CGL) with positive Bcr-ABL. The aim was to prove the effect of the FOXO3a gene polymorphism rs4946936 on the Foxo3a transcription factor. The data analysis test used was a correlation test and a regression test.
 Result : There were three polymorphisms of the FOXO3a gene, namely CC polymorphism, TC polymorphism, and TT polymorphism with a p-value of 0.026 and an r of 0.287, so it can be concluded that there was a significant correlation between the FOXO3a gene polymorphism and the Foxo3an transcription factor with a sufficient correlation value. In the regression test between the FOXO3a gene polymorphisms and the transcription factor FOXO3a, the p value was 0.029 and the B value was -0.294. This means that it has a negative and significant effect on the Foxo3a transcription factor variable.
 Conclusion : There was a significant correlation between the gene polymorphism FOXO3a rs4946936 and the transcription factor FOX3a. The FOXO3a gene polymorphism of the TT genotype had a negative effect on the FOXO3a transcription factor. The TT gene in the FOXO3a gene polymorphism was the most effective in reducing the FOXO3a transcription factor.

FoxO3a Reduces Tamoxifen Resistant Breast Cancer Aggressiveness by Inducing Integrin a5 Expression

Resistance to endocrine therapy is still a major clinical challenge in the management of estrogen receptor positive (ER+) breast cancer (BC) patients, whose poor outcome demands additional studies. Forkhead box class O (FoxO)3a transcription factor, a bona fide oncosuppressor, has been involved in BC metastasis as well as in antiestrogen resistance. Here we demonstrate that the α5 subunit of the integrin α5β1, a well-known membrane heterodimer controlling cell surface adhesion and signaling, is a novel FoxO3a transcriptional target. FoxO3a re-expression reduces motility (wound healing and transmigration assays) of different Tamoxifen resistant BC cell lines, through the induction of α5 mRNA (qRT-PCR) and protein (Western blot) levels. FoxO3a transcriptionally regulates α5 expression by binding to specific Forkhead responsive elements located on the α5 promoter (Luciferase and ChIP assays). Accordingly, using a large-scale BC gene expression datasets from The Cancer Genome Atlas (TCGA) database, a strong positive correlation between FoxO3a and α5 in ER+ BC patients emerged. Altogether, our data unveil an additional mechanism through which FoxO3a activation/induction, by increasing α5 expression, restores a less aggressive phenotype in BC refractory to endocrine therapy.

Senescence associated long non-coding RNA 1 regulates cigarette smoke-induced senescence of type II alveolar epithelial cells through sirtuin-1 signaling

ObjectiveThe primary aim of our study was to explore the mechanisms through which long non-coding RNA (lncRNA)-mediated sirtuin-1 (SIRT1) signaling regulates type II alveolar epithelial cell (AECII) senescence induced by a cigarette smoke-media suspension (CSM).MethodsPharmacological SIRT1 activation was induced using SRT2104 and senescence-associated lncRNA 1 (SAL-RNA1) was overexpressed. The expression of SIRT1, FOXO3a, p53, p21, MMP-9, and TIMP-1 in different groups was detected by qRT-PCR and Western blotting; the activity of SA-β gal was detected by staining; the binding of SIRT1 to FOXO3a and p53 gene transcription promoters was detected by Chip.ResultsWe found that CSM increased AECII senescence, while SAL-RNA1 overexpression and SIRT1 activation significantly decreased levels of AECII senescence induced by CSM. Using chromatin immunoprecipitation, we found that SIRT1 bound differentially to transcriptional complexes on the FOXO3a and p53 promoters.ConclusionOur results suggested that lncRNA-SAL1-mediated SIRT1 signaling reduces senescence of AECIIs induced by CSM. These findings suggest a new therapeutic target to limit the irreversible apoptosis of lung epithelial cells in COPD patients.

Hypermethylation-mediated downregulation of lncRNA PVT1 promotes granulosa cell apoptosis in premature ovarian insufficiency via interacting with Foxo3a.

Long noncoding RNA PVT1 is involved in the progression of female gynecological cancers. However, the role of PVT1 in ovarian granulosa cell apoptosis-mediated premature ovarian insufficiency (POI) remains unclear. This study aims to elucidate the role of PVT1 in ovarian granulosa cell apoptosis-mediated POI. The expression of PVT1 was compared between ovarian tissues from POI patients and normal controls. The methylation level in the PVT1 promoter region was detected by methylation-specific polymerase chain reaction. The interaction between PVT1 and forkhead box class O3A (Foxo3a) was confirmed by RNA pull-down and RNA immunoprecipitation assays. Granulosa cell apoptosis was detected using flow cytometry. The effect of PVT1 on transcription activity of Foxo3a was detected by luciferase reporter assay. The expression of PVT1 was low in the POI ovarian tissues compared with the controls, and such a low expression was related to the hypermethylation of the PVT1 promoter. PVT1 was localized in both the cytoplasm and the nucleus of granulosa cells. We determined that PVT1 could bind with Foxo3a and that downregulating PVT1 by small interfering RNAs inhibited Foxo3a phosphorylation by promoting SCP4-mediated Foxo3a dephosphorylation, resulting in an increase in Foxo3a transcription activity. Moreover, downregulating PVT1 promoted granulosa cell apoptosis by increasing the Foxo3a protein levels. An in vivo experiment showed that the injection of PVT1 overexpressing vectors restored the ovarian function in POI mice. Hypermethylation-induced downregulation of PVT1 promotes granulosa cell apoptosis in POI by inhibiting Foxo3a phosphorylation and increases the Foxo3a transcription activity.

FOXO3a-driven miRNA signatures suppresses VEGF-A/NRP1 signaling and breast cancer metastasis

Metastasis remains the major obstacle to improved survival for breast cancer patients. Downregulation of FOXO3a transcription factor in breast cancer is causally associated with the development of metastasis through poorly understood mechanisms. Here, we report that FOXO3a is functionally related to the inhibition of VEGF-A/NRP1 signaling and to the consequent suppression of breast cancer metastasis. We show that FOXO3a directly induces miR-29b-2 and miR-338 expression. Ectopic expression of miR-29b-2/miR-338 significantly suppresses EMT, migration/invasion, and in vivo metastasis of breast cancer. Moreover, we demonstrate that miR-29b-2 directly targets VEGF-A while miR-338 directly targets NRP1, and show that regulation of miR-29b-2 and miR-338 mediates the ability of FOXO3a to suppress VEGF-A/NRP1 signaling and breast cancer metastasis. Clinically, our results show that the FOXO3a-miR-29b-2/miR-338-VEGF-A/NRP1 axis is dysregulated and plays a critical role in disease progression in breast cancer. Collectively, our findings propose that FOXO3a functions as a metastasis suppressor, and define a novel signaling axis of FOXO3a-miRNA-VEGF-A/NRP1 in breast cancer, which might be potential therapeutic targets for breast cancer.

Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27KIP1 in Conditions Unfavorable for the Growth of Myoblast Cells.

Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated. In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth. For this, we have performed Incucyte® live-cell analysis and applied flow cytometry, western blot, immunoprecipitation, immunohistochemistry, RT-qPCR and luciferase-based methods. Here, we show that lack of CK2α results in significantly delayed cell cycle progression through G1, inhibition of cyclin E-CDK2 complex, decreased phosphorylation of Rb protein at S795, and inactivation of E2F transcription factor. These events are accompanied by nuclear accumulation and up-regulation of the cyclin-dependent kinase inhibitor p27KIP1 in cells and CK2α-knockout mouse tissues. We found that increased levels of p27KIP1 are mainly attributable to post-translational modifications, namely phosphorylation at S10 and T197 amino acid residues catalyzed by Dyrk1B and AMPK, respectively, as silencing of FoxO3A transcription factor, which activates CDKN1B the gene coding for p27KIP1, does not result in markedly decreased expression levels of the corresponding protein. Interestingly, simultaneous silencing of CK2α and p27KIP1 significantly impairs cell cycle progression without increasing cell death. Taken together, our study sheds light on the molecular mechanisms controlling cell cycle progression through G1 phase when myoblasts proliferation potential is impaired by CK2α depletion. Our results suggest that elevated levels of p27KIP1, which follows CK2α depletion, contribute to delay the G1-to-S phase transition. Effects seen when p27KIP1 is down-regulated are independent of CK2α and reflect the protective role exerted by p27KIP1 under unfavorable cell growth conditions.

Abstract A39: Inflammatory macrophage derived TNF alpha induces downregulation of estrogen receptor alpha in breast cancer cells by inactivation of Foxo3A

Abstract Breast cancers are divided into different subtypes based on receptor expression status: estrogen receptor (ER), progesterone receptor (PR) and Her2. The luminal A subtype (ER+PR+Her2-) is most often associated with a good prognosis while the triple-negative (TN) cancers (ER-PR-Her2-) have a poor prognosis. ER+ cancer cells depend on estrogen for their growth and can be treated with drugs to block its effects. While most human breast cancers express ER alpha, some tumors are hormone independent despite the presence of ER and others become ER negative over time. Myeloid cells are known to affect tumor development, and infiltration of tumor-associated macrophages is associated with a worse prognosis. Theoretically, however, macrophages can be both beneficial and bad for the patient. Proinflammatory M1 macrophages (CD68+ CD163-) can eliminate cancer cells and promote antitumor immune responses while anti-inflammatory M2 macrophages (CD163+CD68+) or anti-inflammatory myeloid cells (CD68-CD163+) can promote tumor progression by inhibiting proinflammatory immune responses and induce wound-healing processes. This includes angiogenesis and matrix degradation, both of which promote metastatic spread and possibly even EMT. Macrophages have also been proposed to downregulate ER in breast cancer cells through an unknown mechanism. Due to the recent reports suggesting that macrophages can induce EMT and downregulate ER, we evaluated the effect of human primary macrophages on ER expression in breast cancer cells. Immunohistochemistry analyses of breast cancer tumor xenografts, established with co-transplantation of primary human myeloid cells together with ER+ breast cancer cells in NSG mice, showed association of monocytes with downregulation of ER-receptor expression. Using primary human monocyte-derived macrophage cultures In vitro, we show that the different subtypes of macrophages have unique cytokine secretion profiles with M1 macrophages secreting significantly higher levels of TNF alpha compared to M2 macrophages. ER alpha positive MCF-7 and ER alpha negative MDA-MB-231 breast cancer cells were cultured in macrophage condition media, and both Western blot and qPCR analysis showed downregulation of ER was caused by M1 macrophage-derived TNF alpha that inhibits FoxO3A transcription of ER alpha. We propose that proinflammatory macrophages (M1), despite being tumoricidal, may have direct effects on endocrine resistance in breast cancer patients. Our findings suggest that different macrophage subtypes have various and unique impacts on breast cancer progression, and that proinflammatory macrophages, despite being tumoricidal, may have unwanted and direct effects on endocrine resistance mechanisms in breast cancer patients. Citation Format: Frida Björk Gunnarsdottir, Catharina Hagerling, Caroline Bergenfelz, Camilla M. Rydberg, Meliha Mehmeti, Roni Allaoui, Sofie Mohlin, Sven Påhlman, Daniel Bexell, Karin Leandersson. Inflammatory macrophage derived TNF alpha induces downregulation of estrogen receptor alpha in breast cancer cells by inactivation of Foxo3A [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A39.

C-Jun, Foxo3a, and c-Myc Transcription Factors are Key Regulators of ATP-Mediated Angiogenic Responses in Pulmonary Artery Vasa Vasorum Endothelial Cells.

Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling.

Editor's Note: Sulforaphane Enhances the Therapeutic Potential of TRAIL in Prostate Cancer Orthotopic Model through Regulation of Apoptosis, Metastasis, and Angiogenesis.

Purpose: The purpose of this study was to examine the molecular mechanisms by which sulforaphane enhances the therapeutic potential of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in prostate cancer. Experimental Design: Cell viability and apoptosis assays were done by XTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, respectively. Tumor-bearing mice were treated with vehicle, sulforaphane, TRAIL, and sulforaphane plus TRAIL. Markers of apoptosis, angiogenesis, and metastasis were measured by immunohistochemistry. Results: Sulforaphane enhanced the therapeutic potential of TRAIL in PC-3 cells and sensitized TRAIL-resistant LNCaP cells. Sulforaphane-induced apoptosis in PC-3 cells correlated with the generation of intracellular reactive oxygen species (ROS), collapse of mitochondrial membrane potential, activation of caspase-3 and caspase-9, and up-regulation of DR4 and DR5. Sulforaphane induced the expression of Bax, Bak, Bim, and Noxa and inhibited the expression of Bcl-2, Bcl-X L , and Mcl-1. The quenching of ROS generation with antioxidant N -acetyl-l-cysteine conferred significant protection against sulforaphane-induced ROS generation, mitochondrial membrane potential disruption, caspase-3 activation, and apoptosis. Sulforaphane inhibited growth of orthotopically implanted PC-3 tumors by inducing apoptosis and inhibiting proliferation and also enhanced the antitumor activity of TRAIL. Sulforaphane up-regulated the expressions of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax and Bak and inhibited the activation of nuclear factor-κB P13K/AKT and MEK/ERK pathways in tumor tissues. The combination of sulforaphane and TRAIL was more effective in inhibiting markers of angiogenesis and metastasis and activating FOXO3a transcription factor than single agent alone. Conclusions: The ability of sulforaphane to inhibit tumor growth, metastasis, and angiogenesis and to enhance the therapeutic potential of TRAIL suggests that sulforaphane alone or in combination with TRAIL can be used for the management of prostate cancer.

HIV-1 Vpr inhibits autophagy during the early steps of infection of CD4 Tcells.

Autophagy is induced during HIV-1 entry into CD4 Tcells by the fusion of the membranes triggered by the gp41 envelope glycoprotein. This anti-HIV-1 mechanism is inhibited by the viral infectivity factor (Vif) neosynthesized after HIV-1 integration to allow viral replication. However, autophagy is very rapidly controlled after HIV-1 entry by a still unknown mechanism. As HIV-1 viral protein R (Vpr) is the only auxiliary protein found within the virion in substantial amount, we studied its capability to control the early steps of HIV-1 envelope-mediated autophagy. We demonstrated that ectopic Vpr inhibits autophagy in both the Jurkat CD4 Tcell line and HEK.293Tcells. Interestingly, Vpr coming from the virus also blocks autophagy in CD4 Tcells, the main cell target of HIV-1. Furthermore, Vpr decreases the expression level of two essential autophagy proteins (ATG), LC3B and Beclin-1, and an important autophagy-related protein, BNIP3 as well as the level of their mRNA. We also demonstrated in HEK.293Tcells that Vpr degrades the FOXO3a transcription factor through the ubiquitin proteasome system. Vpr, the only well-expressed HIV-1 auxiliary protein incorporated into viruses, is able to negatively control autophagy induced during HIV-1 entry into CD4 Tcells. We provide insights of how HIV-1 controls autophagy very early after its entry into CD4 Tcells and discovered a new function of Vpr. These results open the route to a better understanding of the roles of Vpr during HIV-1 infection through FOXO3a degradation and could be important to consider additional therapies that counteract the role of Vpr on autophagy.

Corrigendum] The pterocarpanquinone LQB-118 induces apoptosis in acute myeloid leukemia cells of distinct molecular subtypes and targets FoxO3a and FoxM1 transcription factors.

Subsequent to the publication of the above article, the authors have realized that there were errors associated with Figs.1c and2b. In Fig.1c, the authors noted that the same data were incorrectly presented for the 'Untreated cells" and 'DMSO' dot‑blot experiments. After having re‑examined their source data, the authors were able to confirm that the data correctly shown for the 'Untreated cells' experiment had inadvertently been included in the Figure as the data for the 'DMSO' experiment. Additionally, in Fig.2b, the authors noticed that the percentage of untreated cells with active caspase‑3 was missing (the label for the 'No antibody' experiment). Corrected versions of Figs.1 (including the correct data for the 'DMSO' dot blot) and2 (with the label now incorporated) are shown opposite. Note that these changes do not affect the results or the conclusions reported in this paper, and all the authors agree to this correction. The authors apologize to the Editor and to the readership of the Journal for any inconvenience caused. [the original article was published in International Journal of Oncology 45: 1949‑1958, 2014; DOI:10.3892/ijo.2014.2615].