The role of STAT3 in autophagy

Glioblastoma, the most common type of primary malignant brain tumor, is a devastating disease associated with a median survival of approximately 12 months. Here, we have tested E804, the commercially available indirubin derivatives, against U251 and U87 glioblastoma cells. Treatment with E804 significantly inhibits the growth of human glioblastoma cells lines via induction of differentiation and apoptosis. Differentiation induction is coupled with increased expression of glial fibriliary acidic protein, a marker for mature astrocytes. Apoptosis is associated with activation of Caspase 3 and reduction of Bcl-xL and Mcl-1. Furthermore, we demonstrate that E804 reduces signal transducer and activator of transcription-3 (Stat3) signaling to a remarkable extent, suggesting that inactivation of Stat3, at least in part, mediates the effects of this indirubin derivative. Consistently, reduction of Stat3 activity promotes E804-mediated anti-tumor effects, whereas overexpression of Stat3C mutant recues cell apoptosis induced by E804. Taken together, our results indicate that E804 can effectively suppress tumor cell growth, induce tumor cell differentiation and apoptosis mediated partially by Stat3 signaling pathway, suggesting that E804 could be useful for a potential anti-glioblastoma therapeutic approach.

BackgroundSignal transducer and activator of transcription 3 (STAT3) is an oncogene, which upregulates in approximately 70% of human cancers. Autophagy is an evolutionarily conserved process which maintains cellular homeostasis and eliminates damaged cellular components. Moreover, the STAT3 signaling pathway, which may be triggered by cancer cells, has been implicated in the autophagic process.MethodsIn this study, we found that the anthelmintic flubendazole exerts potent antitumor activity in three human colorectal cancer (CRC) cell lines and in the nude mouse model. The inhibition of cell proliferation in vitro by flubendazole was evaluated using a clonogenic assay and the MTT assay. Western blot analysis, flow cytometry analysis, siRNA growth experiment and cytoplasmic and nuclear protein extraction were used to investigate the mechanisms of inhibiting STAT3 signaling and activation of autophagy induced by flubendazole. Additionally, the expression of STAT3 and mTOR was analyzed in paired colorectal cancer and normal tissues collected from clinical patients.ResultsFlubendazole blocked the IL6-induced nuclear translocation of STAT3, which led to inhibition of the transcription of STAT3 target genes, such as MCL1, VEGF and BIRC5. In addition, flubendazole also reduced the expression of P-mTOR, P62, BCL2, and upregulated Beclin1 and LC3-I/II, which are major autophagy-related genes. These processes induced potent cell apoptosis in CRC cells. In addition, flubendazole displayed a synergistic effect with the chemotherapeutic agent 5-fluorouracil in the treatment of CRC.ConclusionsTaken together, these results indicate that flubendazole exerts antitumor activities by blocking STAT3 signaling and inevitably affects the autophagy pathway. Flubendazole maybe a novel anticancer drug and offers a distinctive therapeutic strategy in neoadjuvant chemotherapy of CRC.

LIF is a cytokine playing a key role in the regulation of self-renewal and maintenance of undifferentiated state in mouse ES cells. The response of pluripotent cells to LIF is mediated mainly by the STAT3 and ERK signalling pathways. Recently, we have shown that LIF potentiated retinoic acid-induced neural differentiation of pluripotent mouse embryonal carcinoma P19 cells. Here we demonstrate that pro-neural effects of LIF and partially also of retinoic acid are abolished by inhibition of the JAK2->STAT3 signalling pathway. In contrast, inhibition of the MEK1->ERK signalling pathway does not exhibit any effect. These results suggest that in neurogenic regions, cooperative action of LIF and other neuro-differentiation-inducing factors, such as retinoic acid, may be mediated by the STAT3 signalling pathway.

The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity

Th2 Cytokine-Induced Alterations in Intestinal Smooth Muscle Function Depend on Alternatively Activated Macrophages

BackgroundExosomes play a critical role in the tumor microenvironment by interacting with signaling pathways that facilitate breast cancer metastasis, particularly the STAT3 pathway. The STAT3 pathway is essential for tumor progression and aggressiveness, as it interacts with other pathways like Wnt and Notch in particular, triple-negative breast cancer (TNBC) which is the most aggressive form of breast cancer, with a poor prognosis and rapid metastasis. It is exciting to researchers because it is therapeutically challenging and highly invasive. As a result of the lack of specific treatment options, conventional therapy is widely used, which frequently results in relapse.Objectives Elucidate the critical roles of exosomes in modulating breast cancer behaviour and disease progression in TNBC through STAT3 signaling pathways. Specifically, it was focused on the interplay between STAT3 and Wnt or Notch signalingMaterials and methodsExosomes were isolated from one TNBC patient and characterized using electron microscopy and the western blotting technique. This study utilized two subtypes of breast cancer cell lines: non-TNBC and TNBC. AG490 treatment inhibited STAT3 signaling, and then after inhibition, tumorigenic behaviours were evaluated. Gene expression profiles related to the investigated signaling pathways, Wnt and Notch, were detected.The results and conclusionExosomes significantly affect tumor behaviours and chemoresistance and manipulate signaling pathways associated with tumorigenesis, including Wnt/β-catenin and Notch. These results demonstrated the tumorigenic role of exosomes in the TNBC aggressiveness and suggest that their mechanisms may involve Wnt or Notch signaling mediated by the STAT3.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s12672-025-03334-0.

Direct cardiac reprogramming represents a novel therapeutic strategy to convert non-cardiac cells such as fibroblasts into cardiomyocytes (CMs). This process involves essential transcription factors, such as Mef2c, Gata4, Tbx5 (MGT), MESP1, and MYOCD (MGTMM). However, the small molecules responsible for inducing immature induced CMs (iCMs) and the signaling mechanisms driving their maturation remain elusive. Our study explored the effects of various small molecules on iCM induction and discovered that the combination of FGF4 and ascorbic acid (FA) enhances CM markers, exhibits organized sarcomere and T-tubule structures, and improves cardiac function. Transcriptome analysis emphasized the importance of ECM-integrin-focal adhesions and the upregulation of the JAK2–STAT3 and TGFB signaling pathways in FA-treated iCMs. Notably, JAK2–STAT3 knockdown affected TGFB signaling and the ECM and downregulated mature CM markers in FA-treated iCMs. Our findings underscore the critical role of the JAK2–STAT3 signaling pathway in activating TGFB signaling and ECM synthesis in directly reprogrammed CMs.Schematic showing FA enhances direct cardiac reprogramming and JAK–STAT3 signaling pathways underlying cardiomyocyte maturation.

Spinal cord injury (SCI) is a kind of central nervous system damage that causes significant disability. Although bone marrow stromal cells (BMSCs) have been shown to be an effective therapy for SCI, the regulation mechanism of BMSCs in SCI is unclear. The SCI models were established by oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and laminectomy operation. Exosomes were isolated from the supernatant of BMSC cultures via differential ultracentrifugation, followed by characterization using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and Western blotting. HE and Nissl staining examined pathological changes and neuronal loss in the spinal cord tissues. MTT and PI staining determined cell viability and death. Lipid ROS, iron, GSH, and MDA levels were detected by the C11-BODIPY 581/591 probe and commercial kits. The interactions between p-STAT3 and the TFRC/DMT1 promoter were analyzed by ChIP and dual luciferase reporter gene assay. BMSCs-derived exosomes (BMSCs-exo) suppressed OGD/R-induced neuronal ferroptosis. AKAP12 knockdown weakened the inhibitory impact of BMSCs-exo on OGD/R-induced neuronal ferroptosis. BMSCs-derived exosomal AKAP12 reduced p-ERK and p-STAT3 levels in neuronal cells and p-STAT3 nuclear entry. Additionally, p-STAT3 entering the nucleus increased TFRC and DMT1 promoter transcriptional activity by binding to their promoters. BMSCs-derived exosomal AKAP12 inhibited lipid peroxidation and iron transport signals by inactivating ERK and STAT3 signaling pathways. BMSCs-exo improved the motor function and injury repair in SCI rats by carrying AKAP12. Taken together,BMSCs-derived exosomal AKAP12 inhibited neuronal ferroptosis by repressing TFRC- and DMT1-mediated iron transport signals through inactivating STAT3 and ERK signaling pathways, thereby mitigating SCI. KEY MESSAGES: BMSCs-exo inhibited OGD/R-induced neuronal ferroptosis. AKAP12 silencing in BMSCs weakened the inhibitory impact of BMSCs-exo on OGD/R-induced neuronal ferroptosis. BMSCs-derived exosomal AKAP12 alleviated OGD/R-induced neuronal ferroptosis by inactivating the STAT3 and ERK pathways. Exosomal AKAP12 improved the motor function and injury repair by suppressing the STAT3 and ERK pathways in SCI rats.

Sphingosylphosphorylcholine (SPC) is one of sphingomyelin-derived sphingolipids. SPC levels are increased in ascitic fluids of ovarian cancer patients and stratum corneum of atopic dermatitis (AD) patients. SPC has antitumor activity against several cancer cells by reducing proliferation and migration and increasing apoptosis in vitro. SPC can also cause scratching, potentially exacerbating symptoms of AD. However, the role of SPC in modulating immune responses, particularly in the differentiation of Th9 cells, which carry the most powerful antitumor activity among CD4+ T cells, has yet to be investigated. In this study, we found that SPC is another inducer of Th9 cell differentiation by replicating TGF-β. SPC upregulated Smad3, STAT5, and β-catenin signaling pathways. Increased Smad3 and STAT5 signaling pathways by SPC promoted the differentiation of Th9 cells and increased β-catenin signaling pathway resulted in a less-exhausted, memory-like phenotype of Th9 cells. Increased Smad3, STAT5 and β-catenin signaling pathways by SPC were mediated by increased mitochondrial ROS. These results suggest that SPC is an important endogenous inducer of Th9 cell differentiation and may be one of the targets for treating Th9-related diseases, and that enhancing Th9 differentiation by SPC may be helpful in adoptive T cell therapy for cancer treatment.

Despite being the leading cause of cancer death, targeted therapy for gastric cancer is yet to be established.Wnt/β-catenin signaling is highly deregulated in cancers of gastrointestinal origin including gastric cancers.Stabilization and deregulation of β-catenin occurs at multiple levels and so is being needed to identify a spectrum of Wnt inhibitors to combat deregulated Wnt signaling at the level of various targets and also in different combinations.We developed a luciferase reporter based gastric cancer cellular assay system for Wnt pathway modulator screening and identified nimesulide, a known COX-2 inhibitor as an inhibitor of Wnt/β catenin signaling pathway.Comprehensive signaling pathway profiling revealed that nimesulide could inhibit STAT3, IRF1 and RXR signaling apart from inhibiting Wnt/β-catenin-Myc-E2F signaling cascade.Nimesulide elicits a strong anti-proliferative effect by promoting cell cycle arrest in multiple gastric cancer cell lines.Inhibition of Wnt and STAT3 signaling are found to be COX-2 independent, while the inhibition of RXR and IRF1 pathways are due to the COX-2 inhibiting feature of nimesulide.While nimesulide is capable of activating Notch signaling in gastric cancer cells, celecoxib inhibits Wnt, Myc, E2F, RXR, STAT3, MAPK and Notch signaling pathways in gastric cancer cells.Signaling pathway focused analysis of gastric cancer transcriptome revealed that Wnt, STAT3, IRF1 and RXR signaling pathways are highly deregulated in majority of gastric tumors and indicates the potential of nimesulide and celecoxib class of drugs for targeted gastric cancer therapeutics.The differential inhibition of multiple signaling by nimesulide and celecoxib deserve further investigation.

474 Background: Advanced/metastatic urothelial carcinoma (UC) is a significant public health burden with median overall survival of 15 months. Although, immune checkpoint inhibitors (ICI) have provided an additional second line treatment option, only 15-40% of patients will respond. There has been much effort in determining the mechanisms of immunotherapy resistance and predictive biomarkers to further improve these treatments. Methods: Pre-treatment genomic sequencing data derived from FFPE samples from the IMVIGOR210 clinical trial (n=298) was accessed for analysis. Briefly it was a single arm phase II clinical trial where advanced/metastatic UC patients refractory to platinum chemotherapy treatment received the ICI atezolizumab. This study has been published with detailed methods (PMID: 28950298). The raw sequencing data was pre-processed using standard QC measures and aligned to the human reference genome (hg38). The resulting outputs were then normalized and processed to generate the gene level counts for differential gene expression (DGE). We did DGE analysis comparing patients who had clinical benefit (CR, PR, SD) vs non-clinical benefit (ie. PD) to atezolizumab. The list of differentially expressed genes were then analyzed using various gene ontology, pathway and systems biology tools (IPA, Enrichr, and X2Kweb). Further subset analysis was done using gene-gene correlations (ie. PD-L1 and STAT3) and clinicopathologic features (eg. gender, race, smoking history). Results: Among the 298 patients in this study, there were 25 with CR, 43 with PR, 63 with SD, and 167 with PD based on clinical response to atezolizumab. Subgroup analysis for CR vs PD patients found that approximately 847 genes were differentially expressed with statistical significance (p ≤ 0.05). IPA analysis for this list of differentially expressed genes found among the top signaling pathways were “primary immunodeficiency” and “sirtuin signaling”. Further subset analysis of 39 genes (p ≤ 0.01) enriched in PD patients using Enrichr and X2kweb found that there was an overrepresentation of STAT3 signaling genes (hypergeometric p-val 6.32x104). Conclusions: Our results found that when the transcriptional profiles of CR vs PD there was differential gene expression in STAT3, primary immunodeficiency, and sirtuin signaling pathways. Of note it has been reported that STAT3 signaling can modulate immune activity and its expression is correlated with poor prognosis in urothelial carcinoma patients. These results warrant a larger study to see if STAT3 signaling is a potential biomarker for ICI resistance. If validated this may indicate that the STAT3 pathway is a potential therapeutic target to overcome ICI resistance and improve the efficacy of these agents.

Regulatory effects of noncoding RNAs on the interplay of oxidative stress and autophagy in cancer malignancy and therapy

As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperature, which causes protein misfolding. Misfolded proteins are highly toxic and must be efficiently removed to reduce cellular proteotoxic stress if restoration of native conformations is unsuccessful. Although selective autophagy is known to function in protein quality control by targeting degradation of misfolded and potentially toxic proteins, its role and regulation in heat stress responses have not been analyzed in crop plants. In the present study, we found that heat stress induced expression of autophagy-related (ATG) genes and accumulation of autophagosomes in tomato plants. Virus-induced gene silencing (VIGS) of tomato ATG5 and ATG7 genes resulted in increased sensitivity of tomato plants to heat stress based on both increased development of heat stress symptoms and compromised photosynthetic parameters of heat-stressed leaf tissues. Silencing of tomato homologs for the selective autophagy receptor NBR1, which targets ubiquitinated protein aggregates, also compromised tomato heat tolerance. To better understand the regulation of heat-induced autophagy, we found that silencing of tomato ATG5, ATG7, or NBR1 compromised heat-induced expression of not only the targeted genes but also other autophagy-related genes. Furthermore, we identified two tomato genes encoding proteins highly homologous to Arabidopsis WRKY33 transcription factor, which has been previously shown to interact physically with an autophagy protein. Silencing of tomato WRKY33 genes compromised tomato heat tolerance and reduced heat-induced ATG gene expression and autophagosome accumulation. Based on these results, we propose that heat-induced autophagy in tomato is subject to cooperative regulation by both WRKY33 and ATG proteins and plays a critical role in tomato heat tolerance, mostly likely through selective removal of heat-induced protein aggregates.

BackgroundConstitutive activation of signal transducer and activator of transcription 3 (Stat3) signaling pathway plays an important role in several human cancers. Activation of Stat3 is dependent on the phosphorylation at the tyrosine residue 705 by upstream kinases and subsequent nuclear translocation after dimerization. It remains unclear whether oncogenic Stat3 signaling pathway is involved in the oncogenesis of bladder cancer.ResultsWe found that elevated Stat3 phosphorylation in 19 of 100 (19%) bladder cancer tissues as well as bladder cancer cell lines, WH, UMUC-3 and 253J. To explore whether Stat3 activation is associated with cell growth and survival of bladder cancer, we targeted the Stat3 signaling pathway in bladder cancer cells using an adenovirus-mediated dominant-negative Stat3 (Y705F) and a small molecule compound, STA-21. Both prohibited cell growth and induction of apoptosis in these bladder cancer cell lines but not in normal bladder smooth muscle cell (BdSMC). The survival inhibition might be mediated through apoptotic caspase 3, 8 and 9 pathways. Moreover, down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and survivin) and a cell cycle regulating gene (cyclin D1) was associated with the cell growth inhibition and apoptosis.ConclusionThese results indicated that activation of Stat3 is crucial for bladder cancer cell growth and survival. Therefore, interference of Stat3 signaling pathway emerges as a potential therapeutic approach for bladder cancer.

Myricetin is a type of natural flavonol known for its anticancer activity. However, the molecular mechanism of myricetin in anti-hepatocellular carcinoma (HCC) is not well defined. Previous studies indicated that downregulation of membrane-associated RING-CH finger protein 1 (MARCH1) contributed to the treatment of a variety of cancers. Whether the anticancer property of myricetin is associated with MARCH1 expression remains to be investigated. This research explored the anti-HCC mechanism of myricetin. Our results indicate that myricetin induces autophagy and arrests cell cycle at the G2/M phase to suppress the proliferation of HCC cells by downregulating MARCH1. Myricetin reduces MARCH1 protein in Hep3B and HepG2 cells. Interestingly, myricetin upregulates the MARCH1 mRNA level in Hep3B cells but downregulates it in HepG2 cells. The knockdown of MARCH1 by siRNAs (small interfering RNAs) decreases the phosphorylated p38 MAPK (p-p38 MAPK) and Stat3 (p-Stat3), and inhibits HCC cell viability. Moreover, myricetin inhibits p38 MAPK and Stat3 signaling pathways by downregulating MARCH1 to repress HCC growth both in vitro and in vivo. Bafilomycin A1 (BafA1), an autophagy inhibitor, has synergetic effect with myricetin to inhibit HCC growth. Taken together, our results reveal that myricetin inhibits the proliferation of HCC cells by inhibiting MARCH1-regulated p38 MAPK and Stat3 signaling pathways. This research provides a new molecular mechanism for myricetin in anti-HCC and suggests that targeting MARCH1 could be a novel treatment strategy in developing anticancer therapeutics.