STAT3 Activation Research Articles

Aortic valve stenosis augments long noncoding RNA H19 to govern angiogenic responses and phenotypes of valvular endothelial cell through endothelial to mesenchymal transition

Abstract Background Long noncoding RNAs (lncRNAs) have emerged as important regulators of cardiovascular disease, including aortic valve stenosis (AVS). Although several recent studies have reported that lncRNAs are dysregulated and might play a crucial role in (Endothelial to mesenchymal transition) EndMT, no specific role of lncRNA was reported in AVS pathogenesis. Here, we sought to investigate the role of H19 in EndMT during the pathogenesis of AVS. Methods and results High-throughput lncRNA sequencing of human aortic valve tissue explanted from patients with AVS due to severe stenosis during surgical aortic valve replacement (SAVR) demonstrated that certain lncRNAs [H19, AGAP2-AS1 (PUNISHER), GAS5, MALAT1] are significantly dysregulated compared to patients without AVS. To validate the RNA-seq data, 60 patients with AVS (n=30) or without AVS (n=30) were prospectively investigated. Among these, H19 (p=0.002), AGAP2-AS1 (p=0.002), and GAS5 (0.02) were significantly upregulated in patients with AVS, compared to non-AVS patients. Aortic valvular endothelial cells (VECs) were isolated from explanted valve tissue from AVS patients or obtained commercially. In vitro EndMT-promoting stimuli (TNFα or TGFß+IL1ß) were used for VEC stimulation for 5 days. RT-qPCR was used to quantify the expression of EndMT markers (EC markers, vWF, and NOS3, and mesenchymal markers, αSMA, SM22, Vimentin, and SNAI2). Loss of H19 led to dysregulation of the EC gene network and protein expression patterns that were analyzed by qRT-PCR, immunoblotting, and immunofluorescence, respectively. Further experiments show that silencing of H19 affects migration and proliferation, and promotes EndMT in EC. Functional assays suggested that H19 is a critical regulator of angiogenic properties. The molecular signature of angiogenesis was investigated using a RT-qPCR-based angiogenesis array to identify the regulation of angiogenesis with 84-angiogenesis-related genes. Mechanistically, the angiogenesis array revealed that H19 regulates Jagged Canonical Notch Ligand 1 (JAG1) (4.43-fold vs. control EC), an important regulator of NOTCH1 signaling, and pro-angiogenic factor in EC. Accordingly, EndMT marker expressions were dysregulated upon H19 depletion. A high-throughput luciferase reporter array demonstrated STAT3 activation upon H19 knockdown. Depletion of H19 increased the phosphorylation of STAT3 at TYR705 and pharmacological inhibition of STAT3 activation abolished the effects of H19 silencing on EndMT marker expression as well as on angiogenesis. Overexpression of H19 in VEC abrogated the H19-mediated promotion of JAG1- and VEGFA gene- and protein expression. Our data suggest that H19 might play a role in EC angiogenic gene expression and EndMT. The data reveal a pivotal role of H19 in controlling EndMT in AVS via regulation of the JAG1-NOTCH axis, which could potentially be therapeutically targeted to halt EndMT during AVS.

Thymidine phosphorylase mediates SARS-CoV-2 spike protein enhanced thrombosis in K18-hACE2TG mice

IntroductionThymidine phosphorylase (TYMP), which facilitates platelet activation and thrombosis, is significantly increased in COVID-19 patients. We hypothesize that TYMP mediates SARS-CoV-2 spike protein (SP)-induced thrombosis. Materials and methodsPlasmids encoding wildtype SP or empty vector (p3.1) were transfected into COS-7 cells, and cell lysates were prepared as a reservoir for SP or p3.1 (control), respectively. K18-hACE2TG and K18-hACE2TG/Tymp−/− mice were treated with a single dose of SP or p3.1 by intraperitoneal injection and then subjected to thrombosis studies three days later. The role of SP on inflammatory signaling activation was assessed in BEAS-2B cells. ResultsSARS-CoV-2 SP increased the expression of TYMP, resulting in the activation of STAT3 and NF-κB in BEAS-2B cells. A siRNA-mediated knockdown of TYMP attenuated SP-enhanced activation of STAT3. SP significantly promoted arterial thrombosis in K18-hACE2TG mice. SP-accelerated thrombosis was attenuated by inhibition or genetic ablation of TYMP. SP treatment did not influence ADP- or collagen-induced platelet aggregation but significantly increased platelet adhesion to fibrinogen. SP treatment also significantly shortened activated partial thromboplastin time, which was reversed and even prolonged by TYMP deficiency. Additionally, SP binds to platelet factor 4 (PF4) and TYMP. TYMP does not bind PF4 but enhances the formation of the SP/PF4 complex, which may augment the procoagulant and prothrombotic effect of PF4. ConclusionsWe conclude that SP is prothrombotic and upregulates TYMP expression, and TYMP inhibition or knockout mitigates SP-enhanced thrombosis. These findings suggest that inhibition of TYMP may be a novel therapeutic strategy for COVID-19-associated thrombosis.

Diacerein ameliorates amiodarone-induced pulmonary fibrosis via targeting the TGFβ1/α-SMA/Smad3 pathway.

This study is aimed at investigating the possible protective effect of diacerein (DIA) against AMD-induced pulmonary fibrosis in rats. Rats were classified into 4 groups: a normal group that received distilled water, control group that received AMD (100 mg/kg, p.o.) for 21 days to induce pulmonary fibrosis, and 2 treatment groups that received diacerein, in 2 dose levels (50 and 100 mg/kg, p.o., respectively) in addition to AMD (100 mg/kg, p.o.), for 21 days. Lung function test was assessed using a spirometer; serum and tissue were collected. Biochemical, real-time PCR, histopathological, and immunohistopathological analyses were carried out. AMD reduced tidal volume (TV), peripheral expiratory rate (PER), forced vital capacity (FVC), serum reduced glutathione (GSH) levels, Beclin, and LCII, while it elevated transform growth factor (TGF-β1) gene expression, serum malondialdehyde (MDA) level, alpha-smooth muscle actin (α-SMA), Smad3, phosphorylated signal transducer and activator of transcription (p-STAT3), and p62 lung content. Also, AMD elevated tumor necrosis factor-alpha (TNF-α) and caspase-3 protein expression. DIA elevated TV, PER, FVC, serum GSH level, Beclin, and LCII, while it reduced TGF-β1 gene expression, serum MDA level, α-SMA, Smad3, p-STAT-3, and p62 lung content. Moreover, DIA reduced TNF-α and caspase-3 protein expression. DIA attenuated AMD-induced pulmonary fibrosis via alleviating the TGF1/α-SMA/Smad3 pathway, reducing STAT-3 activation, and combating oxidative stress and inflammation in addition to promoting autophagy and abrogating apoptosis.

Human metapneumovirus SH protein promotes JAK1 degradation to impair host IL-6 signaling.

Human metapneumovirus (HMPV) is a leading cause of respiratory infections in children, older adults, and those with underlying conditions (K. M. Edwards et al., N Engl J Med 368:633-643, 2013, https://doi.org/10.1056/NEJMoa1204630; A. R. Falsey et al., J Infect Dis 187:785-790, 2003, https://doi.org/10.1086/367901; J. S. Kahn, Clin Microbiol Rev 19:546-557, 2006, https://doi.org/10.1128/CMR.00014-06; N. Shafagati and J. Williams, F1000Res 7:135, 2018, https://doi.org/10.12688/f1000research.12625.1). HMPV must evade immune defenses to replicate successfully; however, the viral proteins used to accomplish this are poorly characterized. The HMPV small hydrophobic (SH) protein has been reported to inhibit signaling through type I and type II interferon (IFN) receptors in vitro in part by preventing STAT1 phosphorylation (A. K. Hastings et al., Virology (Auckl) 494:248-256, 2016, https://doi.org/10.1016/j.virol.2016.04.022). HMPV infection also inhibits IL-6 signaling. However, the mechanisms by which SH inhibits signaling and its involvement in IL-6 signaling inhibition are unknown. Here, we used transfection of SH expression plasmids and SH-deleted virus (ΔSH) to show that SH is the viral factor responsible for the inhibition of IL-6 signaling during HMPV infection. Transfection of SH-expression vectors or infection with wild-type, but not ΔSH virus, blocked IL-6-mediated STAT3 activation. Furthermore, JAK1 protein (but not RNA) was significantly reduced in cells infected with wild-type, but not ΔSH virus. The SH-mediated reduction of JAK1 was partially restored by the addition of proteasome inhibitors, suggesting proteasomal degradation of JAK1. Confocal microscopy indicated that infection relocalized JAK1 to viral replication factories. Co-immunoprecipitation showed that SH interacts with JAK1 and ubiquitin, further linking SH to proteasomal degradation machinery. These data indicate that SH inhibits IL-6 and IFN signaling in infected cells in part by promoting proteasomal degradation of JAK1 and that SH is necessary for IL-6 and IFN signaling inhibition in infection. These findings enhance our understanding of the immune evasion mechanisms of an important respiratory pathogen.IMPORTANCEHuman metapneumovirus (HMPV) is a common cause of severe respiratory illness, especially in children and older adults, in whom it is a leading cause of hospitalization. Prior research suggests that severe HMPV infection is driven by a strong immune response to the virus, especially by inflammatory immune signals like interferons (IFN). HMPV produces a small hydrophobic (SH) protein that is known to block IFN signaling, but the mechanism by which it functions and its ability to inhibit other important immune signals remains unexplored. This paper demonstrates that SH can inhibit another related immune signal, IL-6, and that SH depletes JAKs, which are critical proteins involved in both IL-6 and IFN signaling. A robust understanding of how HMPV and related viruses interfere with immune signals important for disease could pave the way for future treatments aimed at mitigating severe infections.

Aripiprazole, but Not Olanzapine, Alters the Response to Oxidative Stress in Fao Cells by Reducing the Activation of Mitogen-Activated Protein Kinases (MAPKs) and Promoting Cell Survival.

Prolonged use of atypical antipsychotics (AAPs) is commonly associated with increased cardiovascular disease risk. While weight gain and related health issues are generally considered the primary contributors to this risk, direct interference with mitochondrial bioenergetics, particularly in the liver where these drugs are metabolized, is emerging as an additional contributing factor. Here, we compared the effects of two AAPs with disparate metabolic profiles on the response of Fao hepatoma cells to oxidative stress: olanzapine (OLA), which is obesogenic, and aripiprazole (ARI), which is not. Results showed that cells treated with ARI exhibited resistance to H2O2-induced oxidative stress, while OLA treatment had the opposite effect. Despite enhanced survival, ARI-treated cells exhibited higher apoptotic rates than OLA-treated cells when exposed to H2O2. Gene expression analysis of pro- and anti-apoptotic factors revealed that ARI-treated cells had a generally blunted response to H2O2, contrasting with a heightened response in OLA-treated cells. This was further supported by the reduced activation of MAPKs and STAT3 in ARI-treated cells in response to H2O2, whereas OLA pre-treatment enhanced their activation. The loss of stress response in ARI-treated cells was consistent with the observed increase in the mitochondrial production of O2•-, a known desensitizing factor. The physiological relevance of O2•- in ARI-treated cells was demonstrated by the increase in mitophagy flux, likely related to mitochondrial damage. Notably, OLA treatment protected proteasome activity in Fao cells exposed to H2O2, possibly due to the better preservation of stress signaling and mitochondrial function. In conclusion, this study highlights the underlying changes in cell physiology and mitochondrial function by AAPs. ARI de-sensitizes Fao cells to stress signaling, while OLA has the opposite effect. These findings contribute to our understanding of the metabolic risks associated with prolonged AAP use and may inform future therapeutic strategies.

KLHL21 suppresses gastric tumourigenesis via maintaining STAT3 signalling equilibrium in stomach homoeostasis

ObjectivePrecancerous metaplasia transition to dysplasia poses a risk for subsequent intestinal-type gastric adenocarcinoma. However, the molecular basis underlying the transformation from metaplastic to cancerous cells remains poorly understood.DesignAn integrated analysis...

Cystathionine-γ-lyase contributes to tamoxifen resistance, and the compound I194496 alleviates this effect by inhibiting the PPARγ/ACSL1/STAT3 signalling pathway in oestrogen receptor-positive breast cancer

Tamoxifen (TAM) resistance is a major challenge in treating oestrogen receptor-positive (ER+) breast cancers. It is possible that the H2S synthase cystathionine-γ-lyase (CSE), which has been previously shown to promote tumour growth and metastasis in other cancer cells, is involved in this resistance. Therefore, we investigated CSE's role and potential mechanisms in TAM-resistant breast cancer cells. First, we examined the effect of CSE expression on TAM sensitivity and resistance in MCF7 (breast cancer) cells. The findings revealed that CSE was directly associated with TAM sensitivity and involved in TAM resistance in ER+ breast cancer cells, indicating that it may be useful as a biomarker. Next, we wanted to determine the molecular mechanism of CSE's role in TAM resistance. Using cell migration, co-immunoprecipitation, western blotting, and cell viability assays, we determined that the CSE/H2S system can affect the expression of PPARγ by promoting the sulfhydrylation of PPARγ, which regulates the transcriptional activity of ACSL1. ACSL1, in turn, influences STAT3 activation by affecting the phosphorylation, palmitoylation and dimerization of STAT3, ultimately leading to the development of TAM resistance in breast cancer. Finally, we examined the effect of CSE inhibitors on reducing drug resistance to determine whether CSE may be used as a biomarker of TAM resistance. We observed that the novel CSE inhibitor I194496 can reverse TAM resistance in TAM-resistant breast cancer via targeting the PPARγ/ACSL1/STAT3 signalling pathway. Overall, our data indicate that CSE may serve as a biomarker of TAM resistance and that the CSE inhibitor I194496 is a promising candidate for combating TAM resistance.

The LINC00319 binding to STAT3 promotes the cell proliferation, migration, invasion and EMT process in oral squamous cell carcinoma

BackgroundLong non-coding RNA LINC00319 has been implicated in the progression of various cancers, including oral squamous cell carcinoma (OSCC). While our previous work has revealed some aspects of LINC00319's role in OSCC, including its upregulation and involvement in a competing endogenous RNA (ceRNA) mechanism, the full extent of its functions and regulatory mechanisms in OSCC progression remain to be fully elucidated. ObjectiveThis study aimed to investigate the function of LINC00319 in OSCC and its potential interaction with the STAT3 signaling pathway, thus uncovering novel regulatory mechanisms and therapeutic targets. MethodsBioinformatics analysis was performed using TCGA data to evaluate LINC00319 expression in OSCC tissues and its correlation with STAT3 signaling. The direct binding between LINC00319 and STAT3 was examined by RNA pull-down, FISH, and RIP assays. Functional experiments, including CCK-8, transwell migration and invasion assays, and western blot analysis of EMT markers and STAT3 pathway activation, were conducted to assess the effects of LINC00319 on OSCC cell behaviors and its interaction with the STAT3 signaling pathway. In vivo xenograft models were established to validate the role of LINC00319 in tumor growth and STAT3 activation. ResultsLINC00319 expression was significantly upregulated in OSCC tissues compared to normal tissues, and high LINC00319 expression correlated with STAT3 signaling activation. Mechanistically, LINC00319 directly bound to STAT3 protein and promoted its phosphorylation at Tyr705. LINC00319 overexpression enhanced, while its knockdown suppressed, the proliferation, migration, invasion, and EMT of OSCC cells. These oncogenic effects were mediated through STAT3 activation and could be reversed by the STAT3 inhibitor stattic. In vivo experiments further confirmed that LINC00319 silencing inhibited tumor growth and STAT3 phosphorylation. ConclusionThis study uncovers that LINC00319 promotes OSCC tumorigenesis by directly binding to and activating STAT3 signaling. These findings provide new insights into the regulatory mechanisms of STAT3 by long non-coding RNAs and highlight the potential of LINC00319 as a biomarker and therapeutic target in OSCC.

Aberrant overexpression of the autoantigen protein vimentin promotes Th17 cell differentiation and autoimmune arthritis via activation of STAT3 signaling

Vimentin contributes to the positioning and function of organelles, cell migration, adhesion, and division. However, secreted vimentin accumulates on the cell surface (Mor-Vaknin et al., 2003; Ramos et al., 2020 [1,2]) where it acts as a coreceptor for viral infection and as an autoantigen in inflammatory and autoimmune diseases. The roles of vimentin in Th17 cells were examined in mice with knockdown of vimentin. We also examined whether STAT3 is required for vimentin expression.Vimentin expression was significantly increased in Th17 cells through STAT3 activation, and vimentin+ IL-17+ T cells were markedly increased in the joint and spleen tissues of CIA mice. The arthritis score and expression levels of proinflammatory cytokines were significantly decreased in CIA mice treated with vimentin shRNA vector.In this study, we demonstrated that vimentin is significantly expressed in Th17 cells through STAT3 activation. Our results provide new insights into the role of vimentin in Th17 cells and the complex pathogenesis of RA.

Detection of heat transfer mechanism in biological systems and HIF-1 α inducing invasion of liver cancer cells in hypoxic environments by activating STAT3

HIF-1α, as a hypoxia-inducing factor, can promote the invasion and metastasis of cancer cells by activating downstream signaling pathways. However, different heat transfer mechanisms in biological systems have important effects on the survival and function of tumor cells. The aim of this study was to investigate how the mechanisms of heat transfer in biological systems affect HIF-1α-mediated STAT3 activation and further induce the invasion ability of liver cancer cells in anoxic environment. The expression and activation of HIF-1α and STAT3 in hepatocellular carcinoma cell lines were detected by establishing anoxic model. At the same time, thermal imaging and heat transfer analysis methods were used to evaluate the heat transfer characteristics of cells under different temperature and hypoxia conditions. The effects of HIF-1α and STAT3 on invasion of hepatocellular carcinoma cells were analyzed in combination with cell migration and invasion experiments. The results showed that the expression of HIF-1α was significantly increased and the STAT3 signaling pathway was activated under hypoxia. The abnormal change of the heat transfer mechanism of biological system accelerates the invasion ability of hepatocellular carcinoma cells. Inhibition of HIF-1α expression can significantly reduce the activation level of STAT3, thereby inhibiting cell migration and invasion. This study reveals the interaction between the biological heat transfer mechanism and HIF-1α-STAT3 signaling pathway under hypoxia environment, which provides a new potential target for the treatment of malignant tumors such as liver cancer, and emphasizes the importance of regulating the heat transfer mechanism.

Diospyros lotus leaf extract and its main component myricitrin inhibit itch‑related IL‑6 and IL‑31 by suppressing microglial inflammation and microglial‑mediated astrocyte activation.

Diospyros lotus has been traditionally used in Asia for medicinal purposes, exhibiting a broad spectrum of pharmacological effects including antioxidant, neuroprotective and anti‑inflammatory properties. While the anti‑itch effect of D. lotus leaves has been reported, studies on the detailed mechanism of action in microglia and astrocytes, which are members of the central nervous system, have yet to be revealed. The present study aimed to investigate effects of D. lotus leaf extract (DLE) and its main component myricitrin (MC) on itch‑related cytokines and signaling pathways in lipopolysaccharide (LPS)‑stimulated microglia. The effect of DLE and MC on activation of astrocyte stimulated by microglia was also examined. Cytokine production was evaluated through reverse transcription PCR and western blot analysis. Signaling pathway was analyzed by performing western blotting and immunofluorescence staining. The effect of microglia on astrocytes activation was evaluated via western blotting for receptors, signaling molecules and itch mediators and confirmed through gene silencing using short interfering RNA. DLE and MC suppressed the production of itch‑related cytokine IL‑6 and IL‑31 in LPS‑stimulated microglia. These inhibitory effects were mediated through the blockade of NF‑κB, MAPK and JAK/STAT pathways. In astrocytes, stimulation by microglia promoted the expression of itch‑related molecules such as oncostatin M receptor, interleukin 31 receptor a, inositol 1,4,5‑trisphosphate receptor 1, lipocalin‑2 (LCN2), STAT3 and glial fibrillary acidic protein. However, DLE and MC significantly inhibited these receptors. Additionally, astrocytes stimulated by microglia with IL‑6, IL‑31, or both genes silenced did not show activation of LCN2 or STAT3. The findings of the present study demonstrated that DLE and MC could suppress pruritic activity in astrocytes induced by microglia‑derived IL‑6 and IL‑31. This suggested the potential of DLE and MC as functional materials capable of alleviating pruritus.

GRB7-mediated enhancement of cell malignant characteristics induced by Helicobacter pylori infection.

Growth factor receptor bound protein 7 (GRB7) is reportedly upregulated in human gastric cancer (GC), which is closely associated with tumor progression and prognosis. However, the mechanism underlying its dysregulation in GC remains poorly understood. In this study, we found that GRB7 overexpression was associated with Helicobacter pylori (H. pylori) infection. GC cells (AGS and MGC-803) infection assays revealed that this upregulation was mediated by the transcription factor STAT3, and activation of STAT3 by H. pylori promoted GRB7 expression in infected GC cells. Moreover, CagA, the key virulence factor of H. pylori, was found involved in STAT3-mediated GRB7 overexpression. The overexpressed GRB7 further promoted GC cell proliferation, migration, and invasion by activating ERK signaling. Mice infection was further used to investigate the action of GRB7. In H. pylori infection, GRB7 expression in mice gastric mucosa was elevated, and higher STAT3 and ERK activation were also detected. These results revealed GRB7-mediated pathogenesis in H. pylori infection, in which H. pylori activates STAT3, leading to increased GRB7 expression, then promotes activation of the ERK signal, and finally enhances malignant properties of infected cells. Our findings elucidate the role of GRB7 in H. pylori-induced gastric disorders, offering new prospects for the treatment and prevention of H. pylori-associated gastric carcinogenesis by targeting GRB7.

Monozygotic triplets with juvenile-onset autoimmunity and 18p microdeletion involving PTPRM.

Abnormal gene dosage from copy number variants has been associated with susceptibility to autoimmune disease. This includes 18p deletion syndrome, a chromosomal disorder with an estimated prevalence of 1 in 50,000 characterized by intellectual disability, facial dysmorphology, and brain abnormalities. The underlying causes for autoimmune manifestations associated with 18p deletions, however, remain unknown. Our objective was to investigate a distinctive case involving monozygotic triplets concordant for developmental delay, white matter abnormalities, and autoimmunity, specifically juvenile-onset Graves' thyroiditis. By chromosomal microarray analysis and whole genome sequencing, we found the triplets to carry a de novo interstitial 5.9Mb deletion of chromosome 18p11.31p11.21 spanning 19 protein-coding genes. We conducted a literature review to pinpoint genes affected by the deletion that could be associated with immune dysregulation and identified PTPRM as a potential candidate. Through dephosphorylation, PTPRM serves as a negative regulator of STAT3, a key factor in the generation of Th17 cells and the onset of specific autoimmune manifestations. We hypothesized that PTPRM hemizygosity results in increased STAT3 activation. We therefore performed assays investigating PTPRM expression, STAT3 phosphorylation, Th1/Th2/Th17 cell fractions, Treg cells, and overall immunophenotype, and in support of the hypothesis, our investigations showed an increase in cells with phosphorylated STAT3 and higher levels of Th17 cells in the triplets. We propose that PTPRM hemizygosity can serve as a contributing factor to autoimmune susceptibility in 18p deletion syndrome. If confirmed in unrelated 18p/PTPRM deletion patients, this susceptibility could potentially be treated by targeted inhibition of IL-17.

Innate immunity gene Nod2 protects mice from orthotopic breast cancer

BackgroundNod2 is involved in innate immune responses to bacteria, regulation of metabolism, and sensitivity to cancer. A Nod2 polymorphism is associated with breast cancer, but the role of Nod2 in the development and progression of breast cancer is unknown.MethodsHere, we tested the hypothesis that Nod2 protects mice from breast cancer using the 4T1 orthotopic model of mammary tumorigenesis. WT and Nod2−/− mice were injected with 4T1 mammary carcinoma cells and the development of tumors was monitored. A detailed analysis of the tumor transcriptome was performed and genes that were differentially expressed and pathways that were predicted to be altered between WT and Nod2−/− mice were identified. The activation of key signaling molecules involved in metabolism and development of cancer was studied.ResultsOur data demonstrate that Nod2−/− mice had a higher incidence and larger tumors than WT mice. Nod2−/− mice had increased expression of genes that promote DNA replication and cell division, and decreased expression of genes required for lipolysis, lipogenesis, and steroid biosynthesis compared with WT mice. Nod2−/− mice also had lower expression of genes required for adipogenesis and reduced levels of lipids compared with WT mice. The tumors in Nod2−/− mice had decreased expression of genes associated with PPARα/γ signaling, increased activation of STAT3, decreased activation of STAT5, and no change in the activation of ERK compared with WT mice.ConclusionsWe conclude that Nod2 protects mice from the 4T1 orthotopic breast tumor, and that tumors in Nod2−/− mice are predicted to have increased DNA replication and cell proliferation and decreased lipid metabolism compared with WT mice.

SPRR1B+ keratinocytes prime oral mucosa for rapid wound healing via STAT3 activation

Oral mucosal wounds exhibit accelerated healing with reduced scarring compared to cutaneous wounds, representing an optimal wound healing paradigm. However, the specific cellular subtypes orchestrating the efficient healing of mucosal tissues remain elusive. Through a comprehensive analysis integrating bulk-mRNA and single-cell sequencing data during the wound healing process in oral mucosa and skin, we have delineated a distinct set of genes markedly upregulated during tissue repair. This collection of wound healing-associated genesets was highly enriched in a specific keratinocyte subpopulation identified as STAT3-activated SPRR1B+ keratinocytes. Notably, despite the inherent rapidity of oral mucosal healing, the induction of SPRR1B+ keratinocytes is evident in both skin and mucosal wound healing processes in murine model. Intriguingly, these wound healing-promoting SPRR1B+ keratinocytes, which are induced via STAT3 activation, inherently abundant in unwounded normal mucosa but absent in normal skin. SPRR1B knockdown significantly inhibits mucosal keratinocyte migration, a critical attribute for effective wound healing. In summary, through analysis of human oral and skin wound healing processes at single-cell resolution, coupled with validation in murine model, suggests STAT3-activated SPRR1B+ keratinocytes are associated with the rapid mucosal repair process. This discovery underscores the potential application of SPRR1B+ keratinocytes in the therapeutic management of chronic or non-healing wounds.

Abstract A060: Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly malignancy. Cancer associated fibroblasts (CAFs) play a complex role in the PDAC tumor microenvironment (TME), with a subset contributing significantly to tumor progression. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss is associated with increased tumor aggressiveness. Our group previously demonstrated PTEN loss in SMA+ positive CAFS correlated with worsened outcomes in patients and in mouse PDAC models. Our group previously demonstrated the loss of PTEN in CAFS led to activation of Stat3, resulting in an immunosuppressive microenvironment (Lefler et al, 2022 PMID: 35803738). However, little is known of the signals produced by tumor cells or other cell types in the TME that trigger loss of PTEN in CAFs. This study aims to to elucidate the molecular mechanisms underlying PTEN loss in CAFs in PDAC. To address this gap, we employed single cell RNA sequencing of tumors from a mouse PDAC model and identified a (PDRGFRa+Lys6+Stat3+Pten-low) CAF subpopulation. Comparing normal pancreas epithelial cells to tumor cells in this data set, we identified the IL6 family of cytokines, (IL6; Oncostatin-M, OSM; and Leukemia Inhibitory Factor, LIF) and Transforming Growth Factor beta 1 (TGFb1) as potential ligands that regulated PTEN, while myeloid cells were a source of the IL6 family member Oncostatin M (OSM). Treatment of isolated CAFs with these factors revealed no significant difference in PTEN mRNA expression. Thus, we developed an in vitro reporter system in which a PTEN-GFP fusion protein is expressed in CAFs. Treating these CAFs with IL6, OSM and TGFb1 (but not LIF) led to a significant decrease in GFP fluorescence. Analysis of endogenous PTEN by western blot confirmed these results. We posited that expression of PTEN E3 ligases might be increased by treatment with these factors, leading to destruction of PTEN. In support of this hypothesis, the expression of E3 ligases WWP1 and WWP2 was increased by treatment with IL6 and OSM. Additionally, preliminary data has revealed that PTEN is oxidized in isolated CAFs, which causes loss of PTEN activity and potentially increased protein turnover. Current experiments are aimed at testing these potential mechanisms. To conclude, PTEN is dysregulated post-transcriptionally in CAFs. The mechanism of PTEN dysregulation could be via increased expression of E3 ligases that target PTEN and/or PTEN oxidation. Future efforts will be aimed at testing whether inhibition of these pathways can restore PTEN expression in mouse PDAC models. These pathways and interactions could be further exploited for therapeutic benefit in curbing PDAC progression. Citation Format: Ivo N. Woogeng, Lu Han, Samaneh Saberi, Cameron Bumbleburg, Joseph Beaudet, Sudarshana Sharma, Michael Ostrowski. Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A060.

Abstract A052: Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy

Abstract Introduction: PDAC is marked by a fibroinflammatory stroma and thrives on interactions between cancer cells and their microenvironment, including CAFs and immune cells, driving disease progression and treatment resistance. Using ChIP-seq on resected human PDAC samples, we identified MICAL2 as a super-enhancer-associated gene and its role in tumor progression. MICAL2 is a flavin monooxygenase that promotes actin depolymerization and SRF transcription. Here, we evaluated how tumor cell-expressed MICAL2 rewires the PDAC microenvironment through the IL1-a/p38 MAP kinase/STAT3 axis. Methods: KPC-Shcontrol (ShCNT) and MICAL2 (M2KD) were orthotopically injected to assess tumor growth. Sc-RNA seq, immunophenotyping, and immunohistochemistry were performed on ShCNT and M2KD tumors. Atomic force microscopy was done to assess tissue stiffness. CD8+ specific antibodies were used for T-cell depletion. Adoptive transfer of CD8+ T cells enriched from M2KD tumors was performed by tail vein injection. Anti-PD1 and anti-IL-1a antibodies were injected IP. RNA-Seq analysis was performed on human PDAC cells (AsPC-1 ShCNT and M2KD) and validation of IL-1α and IL-1R expression was done in human and mouse pancreas control, KD, OE cancer cell lines, and PSCs by q-PCR. We exposed PSCs to conditioned media from cancer cells with and without MICAL2 and checked the expression of genes related to inflammation and fibrosis by qPCR. Results: Orthotopic injections of KPC-M2KD cells led to decreased tumor growth compared to ShCNT (0.26 gm vs. 1 gm, p = 0.008). Sc-RNA and immunohistochemistry revealed reduced PDPN+ and a-SMA+ CAFs in M2KD tumors. We also observed less collagen and fibronectin deposition in M2KD tumors resulting in reduced tissue stiffness as measured by atomic force microscopy. Flow cytometry and immunofluorescence showed increased intertumoral infiltration of cytotoxic and effector CD8+T cells in M2KD tumors. CD8+T cell depletion reversed growth inhibition in M2KD tumors. Adoptive transfer of CD8+T cells enriched from M2KD tumors impeded control tumor growth. RNA-seq revealed decreased IL1-α expression in M2KD cells (p<0.05). IL1R expression was reduced on PSCs when cocultured with M2KD vs. control cell media. PSCs co-cultured with M2KD cancer cells also showed significant downregulation of genes including IL-6, Cxcl1, and LIF changes that were rescued by adding recombinant IL-1α in M2KD cells. Western blot showed MICAL2 regulates the phosphorylation of p38 MAP kinase which in turn regulates the expression of IL1-α and STAT3 activation confirmed by treating dox-inducible ShCNT and ShM2KD cancer cells with p38 inhibitor. Treating M2KD tumors with immune checkpoint blockade PD-1 alone significantly reduced tumor size and 50% of M2KD tumor-bearing mice had complete tumor regression after treatment with PD-1 and neutralizing IL-1a antibodies. Conclusion: MICAL2 mediates tumor-stromal crosstalk through the IL1-a/p38/STAT3 axis and creates an immunosuppressive environment in PDAC. MICAL2 may be a potential immunomodulatory target for pancreatic cancer therapy. Citation Format: Bharti Garg, Evangeline Mose, Edgar Esparaza, Jay Patel, Rithika Medari, Alexei Martsinkovskiy, Carrie Bishop, Gissele Gonzalez, Adam Engler, Parag Katira, Herve Tiriac, Andrew M Lowy. Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A052.

BRUCE liver-deficiency potentiates MASLD/MASH in PTEN liver-deficient background by impairment of mitochondrial metabolism in hepatocytes and activation of STAT3 signaling in hepatic stellate cells.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is currently the most common liver disease, affecting up to 25% of people worldwide, featuring excessive fat accumulation in hepatocytes. Its advanced form, metabolic dysfunction-associated steatohepatitis (MASH), is a serious disease with hepatic inflammation and fibrosis, increasing the need for liver transplants. However, the pathogenic mechanism of MASLD and MASH is not fully understood. We reported that BRUCE ( BIRC6) is a liver cancer suppressor and is downregulated in MASLD/MASH patient liver specimens, though the functional role of BRUCE in MASLD/MASH remains to be elucidated. To this end, we generated liver-specific double KO (DKO) mice of BRUCE and PTEN, a major tumor suppressor and MASLD/MASH suppressor. By comparing liver histopathology among 2-3-month-old mice, there were no signs of MASLD or MASH in BRUCE liver-KO mice and only onset of steatosis in PTEN liver-KO mice. Interestingly, DKO mice had developed robust hepatic steatosis with inflammation and fibrosis. Further analysis of mitochondrial function with primary hepatocytes found moderate reduction of mitochondrial respiration, ATP production and fatty acid oxidation in BRUCE KO and the greatest reduction in DKO hepatocytes. Moreover, aberrant activation of pro-fibrotic STAT3 signaling was found in hepatic stellate cells (HSCs) in DKO mice which was prevented by administered STAT3-specific inhibitor (TTI-101). Collectively, the data demonstrates by maintaining mitochondrial metabolism BRUCE works in concert with PTEN to suppress the pro-fibrogenic STAT3 activation in HSCs and consequentially prevent MASLD/MASH. The findings highlight BRUCE being a new co-suppressor of MASLD/MASH.

Defective N-glycosylation of IL6 induces metastasis and tyrosine kinase inhibitor resistance in lung cancer

The IL6-GP130-STAT3 pathway facilitates lung cancer progression and resistance to tyrosine kinase inhibitors. Although glycosylation alters the stability of GP130, its effect on the ligand IL6 remains unclear. We herein find that N-glycosylated IL6, especially at Asn73, primarily stimulates JAK-STAT3 signaling and prolongs STAT3 phosphorylation, whereas N-glycosylation-defective IL6 (deNG-IL6) induces shortened STAT3 activation and alters the downstream signaling preference for the SRC-YAP-SOX2 axis. This signaling shift induces epithelial-mesenchymal transition (EMT) and migration in vitro and metastasis in vivo, which are suppressed by targeted inhibitors and shRNAs against SRC, YAP, and SOX2. Osimertinib-resistant lung cancer cells secrete a large amount of deNG-IL6 through reduced N-glycosyltransferase gene expression, leading to clear SRC-YAP activation. deNG-IL6 contributes to drug resistance, as confirmed by in silico analysis of cellular and clinical transcriptomes and signal expression in patient specimens. Therefore, the N-glycosylation status of IL6 not only affects cell behaviors but also shows promise in monitoring the dynamics of lung cancer evolution.

Targeting STAT3 signaling pathway by curcumin and its analogues for breast cancer: A narrative review.

Breast cancer (BC) continues to be a significant global health issue, with a rising number of cases requiring ongoing research and innovation in treatment strategies. Curcumin (CUR), a natural compound derived from Curcuma longa, and similar compounds have shown potential in targeting the STAT3 signaling pathway, which plays a crucial role in BC progression. The aim of this study was to investigate the effects of curcumin and its analogues on BC based on cellular and molecular mechanisms. The literature search conducted for this study involved utilizing the Scopus, ScienceDirect, PubMed, and Google Scholar databases in order to identify pertinent articles. This narrative review explores the potential of CUR and similar compounds in inhibiting STAT3 activation, thereby suppressing the proliferation of cancer cells, inducing apoptosis, and inhibiting metastasis. The review demonstrates that CUR directly inhibits the phosphorylation of STAT3, preventing its movement into the nucleus and its ability to bind to DNA, thereby hindering the survival and proliferation of cancer cells. CUR also enhances the effectiveness of other therapeutic agents and modulates the tumor microenvironment by affecting tumor-associated macrophages (TAMs). CUR analogues, such as hydrazinocurcumin (HC), FLLL11, FLLL12, and GO-Y030, show improved bioavailability and potency in inhibiting STAT3, resulting in reduced cell proliferation and increased apoptosis. CUR and its analogues hold promise as effective adjuvant treatments for BC by targeting the STAT3 signaling pathway. These compounds provide new insights into the mechanisms of action of CUR and its potential to enhance the effectiveness of BC therapies.