Inhibition of NLRP3 inflammasome by MCC950 under hypoxia alleviates photoreceptor apoptosis via inducing autophagy in Müller glia.

NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.

STAT3 blockade ameliorates LPS-induced kidney injury through macrophage-driven inflammation.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3), a multifaceted transcription factor, modulates host immune responses by activating cellular response to signaling ligands. STAT3 has a pivotal role in the pathophysiology of kidney injury by counterbalancing resident macrophage phenotypes under inflammation conditions. However, STAT3's role in acute kidney injury (AKI), particularly in macrophage migration, and in chronic kidney disease (CKD) through fibrosis development, remains unclear. METHODS: Stattic (a JAK2/STAT3 inhibitor, 5 mg/kg or 10 mg/kg) was administered to evaluate the therapeutic effect on LPS-induced AKI (L-AKI) and LPS-induced CKD (L-CKD), with animals sacrificed 6-24 h and 14 days post-LPS induction, respectively. The immune mechanisms of STAT3 blockade were determined by comparing the macrophage phenotypes and correlated with renal function parameters. Also, the transcriptomic analysis was used to confirm the anti-inflammatory effect of L-AKI, and the anti-fibrotic role was further evaluated in the L-CKD model. RESULTS: In the L-AKI model, sequential increases in BUN and blood creatinine levels were time-dependent, with a marked elevation of 0-6 h after LPS injection. Notably, two newly identified macrophage subpopulations (CD11bhighF4/80low and CD11blowF4/80high), exhibited population changes, with an increase in the CD11bhighF4/80low population and a decrease in the CD11blowF4/80high macrophages. Corresponding to the FACS results, the tubular injury score, NGAL, F4/80, and p-STAT3 expression in the tubular regions were elevated. STAT3 inhibitor injection in L-AKI and L-CKD mice reduced renal injury and fibrosis. M2-type subpopulation with CD206 in CD11blowF4/80high population increased in the Stattic-treated group compared with that in the LPS-alone group in the L-AKI model. Additionally, STAT3 inhibitor reduced inflammation driven by LPS-stimulated macrophages and epithelial cells injury in the co-culture system. Transcriptomic profiling identified 3 common genes in the JAK-STAT, TLR, and TNF signaling pathways and 11 common genes in the LPS with macrophage response. The PI3K-AKT (IL-6, Akt3, and Pik3r1) and JAK-STAT pathways were determined as potential Stattic targets. Further confirmation through mRNA and protein expressions analyses showed that Stattic treatment reduced inflammation in the L-AKI and fibrosis in the L-CKD mice. CONCLUSIONS: STAT3 blockade effectively mitigated inflammation by retrieving the CD11blowF4/80high population, further emphasizing the role of STAT3-associated macrophage-driven inflammation in kidney injury.

This study investigated the role of STAT3 in LPS-induced acute kidney injury (AKI) and its prolonged pathophysiological effect. In a mouse model, blocking STAT3 with Stattic reduced inflammation and fibrosis, decreased the levels of inflammatory and extracellular matrix (ECM) substances, reduced the number of certain immune cells (macrophages), and influenced specific genes related to inflammation. The findings suggest that targeting STAT3 is a promising approach to treat AKI and CKD by controlling the inflammation and the immune response as well as ECM accumulation. This study provides novel insights into AKI and CKD progression and will facilitate the development of new treatments for kidney injuries at various stages.

Modulation of DOM-Induced Head-Twitch Response by mGluR2 Agonist/Inverse Agonist is Associated with 5-HT2AR-Mediated Gs Signaling Pathway.

Hallucinogenic 5-HT2A receptor (5-HT2AR) agonists-induced head-twitch response (HTR) is regulated by Gs signaling pathway. Formation of heterodimers between 5-HT2AR and metabotropic glutamate mGlu2 receptor (mGluR2) is essential for the hallucinogenic 5-HT2AR agonist-induced HTR. In order to investigate the effects of mGluR2 agonists and inverse agonists on hallucinogenic 5-HT2AR agonists DOM-induced HTR, C57BL/6 mice were pretreated with mGluR2 agonists (LY379268, LY354740, LY404039) or the inverse agonist LY341495, and the HTR was manually counted after administering DOM immediately. IP-One (IP1) HTRF assay and cAMP assay were performed to evaluate the effect of LY341495 or LY354740 on DOM-induced Gq and Gs activation in Human Embryonic Kidney-293 (HEK-293) T-type cells co-expressing 5-HT2AR and mGluR2. The results showed that DOM-induced HTR in mice was dose-dependently inhibited by LY379268, LY354740, and LY404039, while it was dose-dependently enhanced by LY341495. Moreover, LY341495 reversed the inhibitory effect of LY354740 on DOM-induced HTR. In HEK-293T cells co-expressing 5-HT2AR and mGluR2, DOM-induced cAMP level was decreased by LY354740 and increased by LY341495, but DOM-induced IP1 level was not regulated by LY354740 or LY341495. The regulation of DOM-induced HTR by mGluR2 agonists and inverse agonists is closely related to 5-HT2AR-mediated Gs signaling pathway. In HEK-293T cells co-expressing 5-HT2AR and mGluR2 A677S/A681P/A685G mutant (mGluR2 3 A mutant), DOM-induced cAMP level was not regulated by LY354740, but was significantly enhanced by LY341495. The 5-HT2AR/mGluR2 heterodimers is critical for DOM-induced HTR and cAMP level, both of which are inhibited by mGluR2 agonists and enhanced by mGluR2 inverse agonists.

No evidence of clinical efficacy of oxomemazine in cough, according to a systematic review.

PURPOSE: Cough is a prevalent symptom driving patients to seek medical attention in general practice. Despite its widespread use, the clinical efficacy of oxomemazine, the second most reimbursed molecule in France for symptomatic cough treatment, remains uncertain. This study aims to systematically evaluate the clinical efficacy of oxomemazine in cough. METHODS: A systematic literature review with meta-analysis of randomized controlled trials (RCTs) was conducted according to the Rebuild the Evidence Base (REB) protocol. Clinical trials comparing the efficacy of oxomemazine versus placebo or active comparator in cough were searched for. Trials with insufficient data were excluded. Searches were conducted across major databases (Medline, Cochrane Central Register of Controlled Trials, and Embase) and trial registries (World Health Organization International Clinical Trials Registry Platform and ClinicalTrials.gov). RCTs comparing oxomemazine versus placebo or active comparators in cough were sought. Risk of bias was assessed using the Cochrane Collaboration's RoB2 tool. The protocol was preregistered on PROSPERO under the number CRD42022345496 (15). This study received no funding. RESULTS: No RCTs were at low risk of bias. Therefore, no meta-analysis was conducted, in accordance to the pre-specified protocol. CONCLUSIONS: This systematic review highlights the lack of evidence regarding the efficacy of oxomemazine in cough treatment and underscores the need for further well-designed clinical trials to inform its clinical utility in primary care settings.

Neurotoxicity of ischemic astrocytes involves STAT3-mediated metabolic switching and depends on glycogen usage.

Astrocytic responses are critical for the maintenance of neuronal networks in health and disease. In stroke, reactive astrocytes undergo functional changes potentially contributing to secondary neurodegeneration, but the mechanisms of astrocyte-mediated neurotoxicity remain elusive. Here, we investigated metabolic reprogramming in astrocytes following ischemia-reperfusion in vitro, explored their role in synaptic degeneration, and verified the key findings in a mouse model of stroke. Using indirect cocultures of primary mouse astrocytes and neurons, we demonstrate that transcription factor STAT3 controls metabolic switching in ischemic astrocytes promoting lactate-directed glycolysis and hindering mitochondrial function. Upregulation of astrocytic STAT3 signaling associated with nuclear translocation of pyruvate kinase isoform M2 and hypoxia response element activation. Reprogrammed thereby, the ischemic astrocytes induced mitochondrial respiration failure in neurons and triggered glutamatergic synapse loss, which was prevented by inhibiting astrocytic STAT3 signaling with Stattic. The rescuing effect of Stattic relied on the ability of astrocytes to utilize glycogen bodies as an alternative metabolic source supporting mitochondrial function. After focal cerebral ischemia in mice, astrocytic STAT3 activation was associated with secondary synaptic degeneration in the perilesional cortex. Inflammatory preconditioning with LPS increased astrocytic glycogen content, reduced synaptic degeneration, and promoted neuroprotection post stroke. Our data indicate the central role of STAT3 signaling and glycogen usage in reactive astrogliosis and suggest novel targets for restorative stroke therapy.

Prediction of the Renal Organic Anion Transporter 1 (OAT1)- Mediated Drug Interactions for LY404039, the Active Metabolite of Pomaglumetad Methionil.

PURPOSE: The objective of this work was to demonstrate that clinical OAT1-mediated DDIs can be predicted using physiologically based pharmacokinetic (PBPK) modeling. METHODS: LY404039 is a metabotropic glutamate receptor 2/3 agonist and the active moiety of the prodrug pomaglumetad methionil (LY2140023). After oral administration, pomaglumetad methionil is rapidly taken up by enterocytes via PEPT1 and once absorbed, converted to LY404039 via membrane dehydropeptidase 1 (DPEP1). LY404039 is renally excreted by both glomerular filtration and active secretion and in vitro studies showed that the active secretion of LY404039 was mediated by the organic anion transporter 1 (OAT1). Both clinical and in vitro data were used to build a PBPK model to predict OAT1-mediated DDIs. RESULTS: In vitro inhibitory potencies (IC50) of the known OAT inhibitors, probenecid and ibuprofen, were determined to be 4.00 and 2.63 µM, respectively. Subsequently, clinical drug-drug interaction (DDI) study showed probenecid reduced the renal clearance of LY404039 by 30 to 40%. The PBPK bottom-up model, predicted a renal clearance that was approximately 20% lower than the observed one. The middle-out model, using an OAT1 relative activity factor (RAF) of 3, accurately reproduced the renal clearance of LY404039 and pharmacokinetic (PK) changes of LY404039 in the presence of probenecid. CONCLUSIONS: OAT1- mediated DDIs can be predicted using in vitro measured IC50 and PBPK modeling. The effect of ibuprofen was predicted to be minimal (AUC ratio of 1.15) and not clinically relevant.

The STAT3 inhibitor Stattic acts independently of STAT3 to decrease histone acetylation and modulate gene expression.

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in many physiological functions including embryonic development and immune responses and is often activated under pathological conditions such as cancer. Strategies to inactivate STAT3 are being pursued as potential anticancer therapies and have led to the identification of Stattic (6-nitrobenzo[b]thiophene-1,1-dioxide) as a "specific" STAT3 inhibitor that is often used to interrogate STAT3-mediated gene expression in vitro and in vivo. Here, we show that Stattic exerts many STAT3-independent effects on cancer cells, calling for reassessment of results previously ascribed to STAT3 functions. Studies of the STAT3-deficient prostate cancer cell line PC-3 (PC3) along with STAT3-proficient breast cancer cell lines (MDA-MB-231, SUM149) revealed that Stattic attenuated histone acetylation and neutralized effects of the histone deacetylase (HDAC) inhibitor romidepsin. In PC3 cells, Stattic alone inhibited gene expression of CCL20 and CCL2, but activated expression of TNFA, CEBPD, SOX2, and MYC. In addition, we found that Stattic promoted autophagy and caused cell death. These data point to profound epigenetic effects of Stattic that are independent of its function as a STAT3 inhibitor. Our results demonstrate that Stattic directly or indirectly reduces histone acetylation and suggest reevaluation of Stattic and related compounds as polypharmacological agents through multipronged cytotoxic effects on cancer cells.

Randomized Trial of Verubecestat for Prodromal Alzheimer's Disease.

BACKGROUND: Prodromal Alzheimer's disease offers an opportunity to test the effect of drugs that modify the deposition of amyloid in the brain before the onset of dementia. Verubecestat is an orally administered ß-site amyloid precursor protein-cleaving enzyme 1 (BACE-1) inhibitor that blocks production of amyloid-beta (Aß). The drug did not prevent clinical progression in a trial involving patients with mild-to-moderate dementia due to Alzheimer's disease. METHODS: We conducted a randomized, double-blind, placebo-controlled, 104-week trial to evaluate verubecestat at doses of 12 mg and 40 mg per day, as compared with placebo, in patients who had memory impairment and elevated brain amyloid levels but whose condition did not meet the case definition of dementia. The primary outcome was the change from baseline to week 104 in the score on the Clinical Dementia Rating Scale-Sum of Boxes (CDR-SB; scores range from 0 to 18, with higher scores indicating worse cognition and daily function). Secondary outcomes included other assessments of cognition and daily function. RESULTS: The trial was terminated for futility after 1454 patients had been enrolled; 485 had been assigned to receive verubecestat at a dose of 12 mg per day (the 12-mg group), 484 to receive verubecestat at a dose of 40 mg per day (the 40-mg group), and 485 to receive placebo. A total of 234 patients, 231 patients, and 239 patients per group, respectively, completed 104 weeks of the trial regimen. The estimated mean change from baseline to week 104 in the CDR-SB score was 1.65 in the 12-mg group, 2.02 in the 40-mg group, and 1.58 in the placebo group (P = 0.67 for the comparison between the 12-mg group and the placebo group and P = 0.01 for the comparison between the 40-mg group and the placebo group), suggesting a worse outcome in the higher-dose group than in the placebo group. The estimated rate of progression to dementia due to Alzheimer's disease was 24.5, 25.5, and 19.3 events per 100 patient-years in the 12-mg group, the 40-mg group, and the placebo group, respectively (hazard ratio for 40 mg vs. placebo, 1.38; 97.51% confidence interval, 1.07 to 1.79, not adjusted for multiple comparisons), favoring placebo. Adverse events were more common in the verubecestat groups than in the placebo group. CONCLUSIONS: Verubecestat did not improve clinical ratings of dementia among patients with prodromal Alzheimer's disease, and some measures suggested that cognition and daily function were worse among patients who received verubecestat than among those who received placebo. (Funded by Merck Sharp & Dohme; ClinicalTrials.gov number, NCT01953601.).

Neuropathology of a patient with Alzheimer disease treated with low doses of verubecestat.

We report the neuropathological examination of a patient with Alzheimer's disease (AD) treated for 38 months with low doses of the BACE-1 inhibitor verubecestat. Brain examination showed small plaque size, reduced dystrophic neurites around plaques and reduced synaptic-associated Aß compared with a group of age-matched untreated sporadic AD (SAD) cases. Our findings suggest that BACE-1 inhibition has an impact on synaptic soluble Aß accumulation and neuritic derangement in AD.

Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism.

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

Inhibition of STAT3 prevents bone metastatic progression of prostate cancer in vivo.

BACKGROUND: Prostate cancer (PC) metastasizes to the skeleton forming predominantly sclerotic lesions, and there is currently no cure for bone metastatic disease. The transcription factor signal transducer and activator of transcription 3 (STAT3) is implicated as a metastatic driver, but its potential as therapeutic target in bone metastasis has not been investigated. In this study, we evaluated for the first time a STAT3 inhibitor, Napabucasin, as a therapeutic option for bone metastatic PC. METHODS: Effects of STAT3 inhibitors, Stattic and Napabucasin, on metastatic potential in PC cells were studied in vitro by assessment of migration capacity, self-renewal potential, and tumorsphere formation. For evaluation of the role of STAT3 in initial skeletal establishment of PC cells as well as in progressed castration-resistant PC (CRPC) in bone, human VCaP prostate cancer cells were inoculated in the tibia of mice which subsequently were treated with the STAT3 inhibitor Napabucasin. Bone specimens were analyzed using computed tomography (CT), immunohistochemistry, and quantitative polymerase chain reaction. RESULTS: The small molecule STAT3 inhibitors Stattic and Napabucasin both effectively impaired metastatic potential of PC cells in vitro. Furthermore, treatment with Napabucasin prevented metastatic establishment in tibial bones in vivo and thereby also the tumor-induced sclerotic bone response seen in vehicle-treated VCaP xenografts. In addition, treatment with Napabucasin of established bone CRPC significantly decreased both tumor burden and tumor-induced trabecular bone volume compared with effects seen in vehicle-treated animals. Anti-mitotic effects were confirmed by decreased Ki67 staining in Napabucasin-treated xenografts compared with vehicle-treated xenografts. Alterations of gene expression in the femoral bone marrow (BM) niche toward the maintenance of hematopoietic stem cells and the myeloid lineage were demonstrated by quantitative real-time polymerase chain reaction and were further reflected by a substantial increase in the number of erythrocytes in BM of Napabucasin-treated mice. Furthermore, a unique pattern of STAT3 phosphorylation in osteoblasts/stromal cells surrounding the areas of tumor cells was demonstrated immunohistochemically in bone xenograft models using several different PC cell lines. CONCLUSION: Inhibition of STAT3 activity disrupts the bone metastatic niche and targets both the skeletal establishment of PC and advanced bone metastatic CRPC in mice, suggesting STAT3 as a candidate for molecular targeted therapies of skeletal metastatic disease.

Randomized Trial of Verubecestat for Mild-to-Moderate Alzheimer's Disease.

BACKGROUND: Alzheimer's disease is characterized by the deposition of amyloid-beta (Aß) plaques in the brain. Aß is produced from the sequential cleavage of amyloid precursor protein by ß-site amyloid precursor protein-cleaving enzyme 1 (BACE-1) followed by γ-secretase. Verubecestat is an oral BACE-1 inhibitor that reduces the Aß level in the cerebrospinal fluid of patients with Alzheimer's disease. METHODS: We conducted a randomized, double-blind, placebo-controlled, 78-week trial to evaluate verubecestat at doses of 12 mg and 40 mg per day, as compared with placebo, in patients who had a clinical diagnosis of mild-to-moderate Alzheimer's disease. The coprimary outcomes were the change from baseline to week 78 in the score on the cognitive subscale of the Alzheimer's Disease Assessment Scale (ADAS-cog; scores range from 0 to 70, with higher scores indicating worse dementia) and in the score on the Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory scale (ADCS-ADL; scores range from 0 to 78, with lower scores indicating worse function). RESULTS: A total of 1958 patients underwent randomization; 653 were randomly assigned to receive verubecestat at a dose of 12 mg per day (the 12-mg group), 652 to receive verubecestat at a dose of 40 mg per day (the 40-mg group), and 653 to receive matching placebo. The trial was terminated early for futility 50 months after onset, which was within 5 months before its scheduled completion, and after enrollment of the planned 1958 patients was complete. The estimated mean change from baseline to week 78 in the ADAS-cog score was 7.9 in the 12-mg group, 8.0 in the 40-mg group, and 7.7 in the placebo group (P=0.63 for the comparison between the 12-mg group and the placebo group and P=0.46 for the comparison between the 40-mg group and the placebo group). The estimated mean change from baseline to week 78 in the ADCS-ADL score was -8.4 in the 12-mg group, -8.2 in the 40-mg group, and -8.9 in the placebo group (P=0.49 for the comparison between the 12-mg group and the placebo group and P=0.32 for the comparison between the 40-mg group and the placebo group). Adverse events, including rash, falls and injuries, sleep disturbance, suicidal ideation, weight loss, and hair-color change, were more common in the verubecestat groups than in the placebo group. CONCLUSIONS: Verubecestat did not reduce cognitive or functional decline in patients with mild-to-moderate Alzheimer's disease and was associated with treatment-related adverse events. (Funded by Merck; ClinicalTrials.gov number, NCT01739348 .).

Signal transducer and activator of transcription 3 inhibition alleviates resistance to BRAF inhibition in anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is a rare type of thyroid cancer (TC) with no effective therapeutic strategy. Although surgery, chemotherapy and radiation are all available for ATC treatment, the median survival for ATC patients is less than 6 months. In this study, we aimed to study on resistant mechanisms to B-Raf proto-oncogene serine/threonine kinase (BRAF) inhibitor and identify effective combinational therapy for ATC patients. TC cells were treated with Vemurafenib and cell apoptosis and viability were analyzed by flow cytometry and MTT assay. Monolayer and sphere cells were isolated from ATC cells to detect the mRNA level of stem cell markers and differentiation markers by RT-PCR. Phosphor-STAT3 level in sphere and monolayer cells was tested by Western blotting. The xenotransplantation animal model has established to analyze the anti-tumor effect of Vemurafenib and Stattic combinational therapy. Undifferentiated TC cells were resistant to Vemurafenib treatment. Sphere cells isolated from ATC showed no significant change in cell viability and apoptosis upon Vemurafenib treatment, and expressed a high level of stem cell marker and phosphor-STAT3. STAT3 inhibition enhanced the tumorigenic capacity and increased Vemurafenib sensitivity in ATC cell lines. Stattic significantly enhanced anti-tumor effect of Vemurafenib in mouse model. Our findings demonstrate that the combinational therapy of Vemurafenib and Stattic is an effective therapeutic treatment for ATC patients.

Inhibition of STAT3 enhances sensitivity to tamoxifen in tamoxifen-resistant breast cancer cells.

BACKGROUND: The mechanisms of endocrine resistance are complex, and deregulation of several oncogenic signalling pathways has been proposed. We aimed to investigate the role of the EGFR and Src-mediated STAT3 signalling pathway in tamoxifen-resistant breast cancer cells. METHODS: The ER-positive luminal breast cancer cell lines, MCF-7 and T47D, were used. We have established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with 4-hydroxytamoxifen. Cell viability was determined using an MTT assay, and protein expression levels were determined using western blot. Cell cycle and annexin V staining were analysed using flow cytometry. RESULTS: TamR cells showed decreased expression of estrogen receptor and increased expression of EGFR. TamR cells showed an acceleration of the G1 to S phase transition. The protein expression levels of phosphorylated Src, EGFR (Y845), and STAT3 was increased in TamR cells, while phosphorylated Akt was decreased. The expression of p-STAT3 was enhanced according to exposure time of tamoxifen in T47D cells, suggesting that activation of STAT3 can cause tamoxifen resistance in ER-positive breast cancer cells. Both dasatinib (Src inhibitor) and stattic (STAT3 inhibitor) inhibited cell proliferation and induced apoptosis in TamR cells. However, stattic showed a much stronger effect than dasatinib. Knockdown of STAT3 expression by siRNA had no effect on sensitivity to tamoxifen in MCF-7 cells, while that enhanced sensitivity to tamoxifen in TamR cells. There was not a significant synergistic effect of dasatinib and stattic on cell survival. TamR cells have low nuclear p21(Cip1) expression compared to MCF-7 cells and inhibition of STAT3 increased the expression of nuclear p21(Cip1) in TamR cells. CONCLUSIONS: The EGFR and Src-mediated STAT3 signalling pathway is activated in TamR cells, and inhibition of STAT3 may be a potential target in tamoxifen-resistant breast cancer. An increase in nuclear p21(Cip1) may be a key step in STAT3 inhibitor-induced cell death in TamR cells.

Macrophages Promote Aortic Valve Cell Calcification and Alter STAT3 Splicing.

OBJECTIVE: Macrophages have been described in calcific aortic valve disease, but it is unclear if they promote or counteract calcification. We aimed to determine how macrophages are involved in calcification using the Notch1+/- model of calcific aortic valve disease. Approach and Results: Macrophages in wild-type and Notch1+/- murine aortic valves were characterized by flow cytometry. Macrophages in Notch1+/- aortic valves had increased expression of MHCII (major histocompatibility complex II). We then used bone marrow transplants to test if differences in Notch1+/- macrophages drive disease. Notch1+/- mice had increased valve thickness, macrophage infiltration, and proinflammatory macrophage maturation regardless of transplanted bone marrow genotype. In vitro approaches confirm that Notch1+/- aortic valve cells promote macrophage invasion as quantified by migration index and proinflammatory phenotypes as quantified by Ly6C and CCR2 positivity independent of macrophage genotype. Finally, we found that macrophage interaction with aortic valve cells promotes osteogenic, but not dystrophic, calcification and decreases abundance of the STAT3ß isoform. CONCLUSIONS: This study reveals that Notch1+/- aortic valve disease involves increased macrophage recruitment and maturation driven by altered aortic valve cell secretion, and that increased macrophage recruitment promotes osteogenic calcification and alters STAT3 splicing. Further investigation of STAT3 and macrophage-driven inflammation as therapeutic targets in calcific aortic valve disease is warranted.

BACE inhibition causes rapid, regional, and non-progressive volume reduction in Alzheimer's disease brain.

In the phase 3 EPOCH trial (Clinicaltrials.gov; NCT01739348), treatment with the BACE inhibitor verubecestat failed to improve cognition in patients with mild-to-moderate Alzheimer's disease, but was associated with reduced hippocampal volume after 78 weeks as assessed by MRI. The aims of the present exploratory analyses were to: (i) characterize the effect of verubecestat on brain volume by evaluating the time course of volumetric MRI changes for a variety of brain regions; and (ii) understand the mechanism through which verubecestat might cause hippocampal (and other brain region) volume loss by assessing its relationship to measures of amyloid, neurodegeneration, and cognition. Participants were aged 55-85 years with probable Alzheimer's disease dementia and a Mini Mental State Examination score ≥15 and ≤26. MRIs were obtained at baseline and at Weeks 13, 26, 52 and 78 of treatment. MRIs were segmented using Freesurfer and analysed using a tensor-based morphometry method. PET amyloid data were obtained with 18F-flutemetamol (Vizamyl®) at baseline and Week 78. Standardized uptake value ratios were generated with subcortical white matter as a reference region. Neurofilament light chain in the CSF was assessed as a biomarker of neurodegeneration. Compared with placebo, verubecestat showed increased MRI brain volume loss at Week 13 with no evidence of additional loss through Week 78. The verubecestat-related volumetric MRI loss occurred predominantly in amyloid-rich brain regions. Correlations between amyloid burden at baseline and verubecestat-related volumetric MRI reductions were not significant (r = 0.05 to 0.26, P-values > 0.27). There were no significant differences between verubecestat and placebo in changes from baseline in CSF levels of neurofilament light chain at Week 78 (increases of 7.2 and 14.6 pg/ml for verubecestat versus 19.7 pg/ml for placebo, P-values ≥ 0.1). There was a moderate correlation between volumetric MRI changes and cognitive decline in all groups including placebo at Week 78 (e.g. r = -0.45 to -0.55, P < 0.001 for whole brain), but the correlations were smaller at Week 13 and significant only for the verubecestat groups (e.g. r = -0.15 and -0.11, P < 0.04 for whole brain). Our results suggest that the verubecestat-associated MRI brain volume loss is not due to generalized, progressive neurodegeneration, but may be mediated by specific effects on BACE-related amyloid processes.

Stattic sensitizes osteosarcoma cells to epidermal growth factor receptor inhibitors via blocking the interleukin 6-induced STAT3 pathway.

Osteosarcoma (OS), the most common malignant bone tumor with high metastatic potential, frequently affects children and adolescents. Epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors exhibit encouraging anti-tumor activity for patients with solid tumors, whereas their effects on OS remain controversial. In the present study, we aimed to elucidate the anti-tumor activity of gefitinib for OS, as well as to explore the underlying mechanisms. Gefitinib inhibits cell viability, tumor growth, cell migration, and invasion and promotes cell apoptosis and G1 cycle arrest in OS at a relatively high concentration via suppressing the PI3K/Akt and ERK pathways. However, gefitinib treatment results in the feedback activation of signal transducer and activator of transcription 3 (STAT3) induced by interleukin 6 (IL-6) secretion. Combined treatment with gefitinib and stattic, an inhibitor for STAT3 phosphorylation, engenders more evident inhibitory effects on cell proliferation, migration, and invasion and promotive effects on cell apoptosis and G1 phase arrest in OS, compared with the single exposure to gefitinib or stattic. Western blot analysis demonstrates that stattic treatment in gefitinib-treated OS abrogates the IL-6-induced STAT3 activation and subsequently further restrains the activities of EGFR, Akt, and ERK pathways in tumor cells. This study confirms that the EGFR inhibitor of gefitinib has moderate anti-tumor effects on OS through IL-6 secretion-mediated STAT3 activation. Additional administration of stattic in EGFR-targeted therapies may contribute to improve the efficacy for OS.

Hiltonol Cocktail Kills Lung Cancer Cells by Activating Cancer-Suppressors, PKR/OAS, and Restraining the Tumor Microenvironment.

NSCLC (non-small cell lung cancer) is a leading cause of cancer-related deaths worldwide. Clinical trials showed that Hiltonol, a stable dsRNA representing an advanced form of polyI:C (polyinosinic-polycytidilic acid), is an adjuvant cancer-immunomodulator. However, its mechanisms of action and effect on lung cancer have not been explored pre-clinically. Here, we examined, for the first time, how a novel Hiltonol cocktail kills NSCLC cells. By retrospective analysis of NSCLC patient tissues obtained from the tumor biobank; pre-clinical studies with Hiltonol alone or Hiltonol+++ cocktail [Hiltonol+anti-IL6+AG490 (JAK2 inhibitor)+Stattic (STAT3 inhibitor)]; cytokine analysis; gene knockdown and gain/loss-of-function studies, we uncovered the mechanisms of action of Hiltonol+++. We demonstrated that Hiltonol+++ kills the cancer cells and suppresses the metastatic potential of NSCLC through: (i) upregulation of pro-apoptotic Caspase-9 and Caspase-3, (ii) induction of cytosolic cytochrome c, (iii) modulation of pro-inflammatory cytokines (GRO, MCP-1, IL-8, and IL-6) and anticancer IL-24 in NSCLC subtypes, and (iv) upregulation of tumor suppressors, PKR (protein kinase R) and OAS (2'5' oligoadenylate synthetase). In silico analysis showed that Lys296 of PKR and Lys66 of OAS interact with Hiltonol. These Lys residues are purportedly involved in the catalytic/signaling activity of the tumor suppressors. Furthermore, knockdown of PKR/OAS abrogated the anticancer action of Hiltonol, provoking survival of cancer cells. Ex vivo analysis of NSCLC patient tissues corroborated that loss of PKR and OAS is associated with cancer advancement. Altogether, our findings unraveled the significance of studying tumor biobank tissues, which suggests PKR and OAS as precision oncological suppressor candidates to be targeted by this novel Hiltonol+++ cocktail which represents a prospective drug for development into a potent and tailored therapy for NSCLC subtypes.

ML264 inhibits osteosarcoma growth and metastasis via inhibition of JAK2/STAT3 and WNT/ß-catenin signalling pathways.

Osteosarcoma, the most common bone malignancy, has a high morbidity rate and poor prognosis. Krüppel-like factor 5 (KLF5) is a key transcriptional regulator of cellular proliferation whose overexpression is observed in osteosarcoma cell lines (U2OS, 143B, MG63 and SAOS2). ML264, a small-molecule inhibitor of KLF5, exerts antiproliferative effects in colorectal cancer; however, its function in osteosarcoma remains unknown. Here, we explored the possible antitumour effects of ML264 on 143B and U2OS cell lines and murine tumour xenograft model. ML264 suppressed proliferation and clonogenic ability of osteosarcoma cells in a dose-dependent manner. Moreover, ML264 induced G0/G1 cell cycle arrest, with no influence on apoptosis, and inhibited the migratory and invasive abilities of osteosarcoma cells, as demonstrated by wound-healing and Transwell assays. Exposure to ML264 reduced the mRNA and protein levels of molecules associated with epithelial-mesenchymal transition phenotype, including N-cadherin, vimentin, Snail, matrix metalloproteinase (MMP) 9 and MMP13. Inhibition of signal transducer and activator of transcription (STAT) 3 phosphorylation and Wnt signalling was also observed. In the murine model of osteosarcoma, tumour growth was efficiently suppressed following a 10-day treatment with ML264. Collectively, our findings demonstrate the potential value of ML264 as a novel anticancer drug for osteosarcoma.

Dual PLK1 and STAT3 inhibition promotes glioblastoma cells apoptosis through MYC.

Glioblastoma (GBM) is the deadliest primary brain tumor that is highly resistant to current treatments. Polo-like kinase 1 (PLK1) and signal transducer and activator of transcription 3 (STAT3) are highly expressed in gliomas, especially GBM. Previous studies have shown reciprocal activation between PLK1 and STAT3 and that they regulate the same pools of MYC downstream. We have demonstrated that PLK1 and STAT3 levels are elevated in gliomas compared with those in normal brain tissues, and high expression of both PLK1 and STAT3 is associated with poor prognosis in TCGA. Moreover, there was direct or indirect reciprocal regulation between PLK1 and STAT3. Furthermore, we found that PLK1 and STAT3 can regulate the same pools of MYC downstream. Compared to monotherapy, combined treatment of glioma cells with PLK1 and STAT3 inhibitors, BI2536 and Stattic, respectively, showed lower expression of MYC, synergistic induction of cell invasion and apoptosis in vitro, and tumor inhibition in xenografts. PLK1 and STAT3 were able to directly regulate the expression of MYC and induce apoptosis of glioma cells through the regulation of MYC. These findings may help develop a therapeutic strategy for dual inhibition of PLK1 and STAT3 against the tumorigenesis of glioma.