Signal Transducer And Activator Of Transcription 3 Activation Research Articles

Abstract PO1-25-08: Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer

Abstract Background: The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase overexpressed in approximately 20-25% of breast cancers with varying biological differences according to hormone receptor (HR) positive status. Combination of chemotherapy with dual HER2 blockade and endocrine therapy (adjuvant) or CDK4/6 inhibitor (CDK4/6i) therapy (metastatic) is standard of care for HER2-positive/HR-positive breast cancer. However, de-escalation of chemotherapy remains of significant interest given associated toxicities and, as a result, alternative therapeutic strategies are needed. One such strategy is targeting the signal transducer and activator of transcription 3 (STAT3) which is constitutively activated in the HER-2 positive setting. Moreover, there is preclinical evidence to suggest that phosphorylated STAT3 (pSTAT3) induces overexpression of cyclin D1 (CCND1), which along with CDK4 may mediate resistance to targeted therapy for HER2-positive breast cancer. However, it is unclear if pSTAT3 plays a significant role in tumorigenesis or the development of resistance to therapy in HER2-positive disease. We aim to evaluate the effect of CDK4/6i therapy with palbociclib on tumor growth in an inducible transgenic HER2-positive mouse model and the impact on pSTAT3 and cyclin D1 levels. Methods: Using an inducible transgenic mouse mammary tumor virus (MMTV) model of HER2-positive breast cancer, we evaluated STAT3 phosphorylation in tumors driven by HER2 expression in addition to the impact of CDK4/6 inhibition with palbociclib on tumorigenesis and pSTAT3 activation. To model tumor recurrence, doxycycline was withdrawn from tumor bearing mice and tumor regression was observed. Subsequently, the mice developed recurrent mammary tumors. Given recurrent tumors in this model are driven by a HER2-independent mechanism, we examined expression of cell cycle proteins together with STAT3 phosphorylation to determine association with recurrence. Result: In addition to expressing HER2, the primary tumors expressed estrogen receptor (ERα), Rb, cyclin D1, and CDK4/6 proteins. STAT3 phosphorylation was also observed. Treatment with palbociclib decreased Rb phosphorylation and decreased tumor growth compared to vehicle treated mice (%TGI 79.37% after 21 days). Furthermore, pSTAT3 levels were not altered with palbociclib therapy. When recurrent tumors were analyzed, they lacked HER2 expression but expressed cyclin D1 and CDK4/6 supporting that tumor growth is driven by cyclin D1/CDK4 pathway. Recurrent tumors also demonstrated pSTAT3 despite lack of HER2 expression suggesting that STAT3 phosphorylation in the recurrent tumors is mediated by an alternative mechanism, potentially the CDK4/6 pathway. Conclusion: We demonstrate STAT3 activation in a mouse model of HER2 driven breast cancer and show persistent activation in recurrent tumors lacking HER2 expression rendering them resistant to HER2-targeted therapy. We also demonstrate the anti-tumor effect of palbociclib in primary tumors with HER2-dependent proliferation. Taken together, our findings suggest that pSTAT3 is a viable therapeutic target and provides the rationale for combination therapy with CDK4/6 inhibition using palbociclib and STAT3 inhibition with a novel STAT3 inhibitor in HER2-positive breast cancer. This therapeutic approach may represent a potential chemotherapy de-escalation strategy. Citation Format: Taiwo Adesoye, Linjie Luo, Tuyen Bui, Serena Kim, Hannah Wingate, Debu Tripathy, Kelly Hunt, Khandan Keyomarsi. Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-08.

Spinal lipocalin 2 as a factor in the development of central post-stroke pain.

Central poststroke pain (CPSP) is a type of central neuropathic pain whose mechanisms remain unknown. Recently, we showed that activated astrocytes and microglial cells are present in the spinal cord of CPSP model mice. Activated glial cells exacerbate cerebral ischemic pathology by increasing the expression of inflammatory factors. However, the involvement of spinal glial cells in CPSP remains unknown. We hypothesized that spinal glial cell-derived molecules cause hyperexcitability or promoted the development of CPSP. In this study, we identified glial cell-derived factors involved in the development of CPSP using a bilateral common carotid occlusion (BCAO)-induced CPSP mouse model. Male ddY mice were subjected to BCAO for 30 min. The von Frey test assessed mechanical hypersensitivity in the right hind paw of mice. BCAO mice showed hypersensitivity to mechanical stimuli and astrocyte activation in the spinal cord 3 days after treatment. DNA microarray analysis revealed a significant increase in lipocalin 2 (LCN2), is known as neutrophil gelatinase-associated lipocalin, in the superficial dorsal horns of BCAO-induced CPSP model mice. LCN2 colocalized with GFAP, an astrocyte marker. Spinal GFAP-positive cells in BCAO mice co-expressed signal transducer and activator of transcription 3 (STAT3). The increase in the fluorescence intensity of LCN2 and GFAP in BCAO mice was suppressed by intrathecal injection of AG490, an inhibitor of JAK2 and downstream STAT3 activation, or anti-LCN2 antibody. Our findings indicated that LCN2 in spinal astrocytes may be a key molecule and may be partly involved in the development of CPSP.

The endonuclease FEN1 mediates activation of STAT3 and facilitates proliferation and metastasis in breast cancer.

The metastasis accounts for most deaths from breast cancer (BRCA). Understanding the molecular mechanisms of BRCA metastasis is urgently demanded. Flap Endonuclease 1 (FEN1), a pivotal factor in DNA metabolic pathways, contributes to tumor growth and drug resistance, however, little is known about the role of FEN1 in BRCA metastasis. In this study, FEN1 expression and its clinical correlation in BRCA were investigated using bioinformatics, showing being upregulated in BRCA samples and significant relationships with tumor stage, node metastasis, and prognosis. Immunohistochemistry (IHC) staining of local BRCA cohort indicated that the ratio of high FEN1 expression in metastatic BRCA tissues rose over that in non-metastatic tissues. The assays of loss-of-function and gain-of-function showed that FEN1 enhanced BRCA cell proliferation, migration, invasion, xenograft growth as well as lung metastasis. It was further found that FEN1 promoted the aggressive behaviors of BRCA cells via Signal Transducer and Activator of Transcription 3 (STAT3) activation. Specifically, the STAT3 inhibitor Stattic thwarted the FEN1-induced enhancement of migration and invasion, while the activator IL-6 rescued the decreased migration and invasion caused by FEN1 knockdown. Additionally, overexpression of FEN1 rescued the inhibitory effect of nuclear factor-κB (NF-κB) inhibitor BAY117082 on phosphorylated STAT3. Simultaneously, the knockdown of FEN1 attenuated the phosphorylation of STAT3 promoted by the NF-κB activator tumor necrosis factor α (TNF-α). These results indicate a novel mechanism that NF-κB-driven FEN1 contributes to promoting BRCA growth and metastasis by STAT3 activation.

Novel STAT3 oligonucleotide compounds suppress tumor growth and overcome the acquired resistance to sorafenib in hepatocellular carcinoma.

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the occurrence and progression of tumors, leading to resistance and poor prognosis. Activation of STAT3 signaling is frequently detected in hepatocellular carcinoma (HCC), but potent and less toxic STAT3 inhibitors have not been discovered. Here, based on antisense technology, we designed a series of stabilized modified antisense oligonucleotides targeting STAT3 mRNA (STAT3 ASOs). Treatment with STAT3 ASOs decreased the STAT3 mRNA and protein levels in HCC cells. STAT3 ASOs significantly inhibited the proliferation, survival, migration, and invasion of cancer cells by specifically perturbing STAT3 signaling. Treatment with STAT3 ASOs decreased the tumor burden in an HCC xenograft model. Moreover, aberrant STAT3 signaling activation is one of multiple signaling pathways involved in sorafenib resistance in HCC. STAT3 ASOs effectively sensitized resistant HCC cell lines to sorafenib in vitro and improved the inhibitory potency of sorafenib in a resistant HCC xenograft model. The developed STAT3 ASOs enrich the tools capable of targeting STAT3 and modulating STAT3 activity, serve as a promising strategy for treating HCC and other STAT3-addicted tumors, and alleviate the acquired resistance to sorafenib in HCC patients. A series of novel STAT3 antisense oligonucleotide were designed and showed potent anti-cancer efficacy in hepatocellular carcinoma in vitro and in vivo by targeting STAT3 signaling. Moreover, the selected STAT3 ASOs enhance sorafenib sensitivity in resistant cell model and xenograft model.

Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.

LINC01686 affects LPS-induced cytokine expression via the miR-18a-5p/A20/STAT1 axis in THP-1 cells.

Long noncoding RNAs (lncRNAs) are crucial in regulating various physiological and pathological processes, including immune responses. LINC01686 is a lncRNA with previously uncharacterized functions in immune regulation. This study aims to investigate the function of LINC01686 in lipopolysaccharide (LPS)-induced inflammatory responses in the human monocytic leukemia cell line THP-1 and its potential regulatory mechanisms involving miR-18a-5p and the anti-inflammatory protein A20. THP-1 cells were stimulated with LPS to induce inflammatory responses, followed by analysis of LINC01686 expression levels. The role of LINC01686 in regulating the expression of interleukin (IL)-6, IL-8, A20, and signal transducer and activator of transcription 1 (STAT1) was examined using small interfering RNA-mediated knockdown. Additionally, the involvement of miR-18a-5p in LINC01686-mediated regulatory pathways was assessed by transfection with decoy RNAs mimicking the miR-18a-5p binding sites of LINC01686 or A20 messenger RNA. LINC01686 expression was upregulated in THP-1 cells following LPS stimulation. Suppression of LINC01686 enhanced LPS-induced expression of IL-6 and IL-8, mediated through increased production of reactive oxygen species. Moreover, LINC01686 knockdown upregulated the expression and activation of IκB-ζ, STAT1, and downregulated A20 expression. Transfection with decoy RNAs reversed the effects of LINC01686 suppression on A20, STAT1, IL-6, and IL-8 expression, highlighting the role of LINC01686 in sponging miR-18a-5p and regulating A20 expression. This study provides the first evidence that LINC01686 plays a critical role in modulating LPS-induced inflammatory responses in THP-1 cells by sponging miR-18a-5p, thereby regulating the expression and activation of A20 and STAT1. These findings shed light on the complex regulatory mechanisms involving lncRNAs in immune responses and offer potential therapeutic targets for inflammatory diseases.

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer

BackgroundOvarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient...

Inhibition of complement C3 prevents osteoarthritis progression in guinea pigs by blocking STAT1 activation

Osteoarthritis (OA) is one of the leading causes of disability, affecting over 500 million adults worldwide. Previous studies have found that various inflammatory factors can contribute to the pathogenesis of OA, including complement factors in the synovial fluid of OA patients. However, the pathogenesis of this disease is still not known, and the only therapy of severe OA is total joint replacements. Total joint replacements are invasive, expensive, and affect quality of life. Here we show that when human articular chondrocytes are stimulated with pro-inflammatory mediator interleukin-1β (IL-1β) there is an increase in inflammatory factors including complement component 3 (C3). We also found the transcription factor, signal transducer and activator of transcription 1 (STAT1), is responsible for increased C3 expression after IL-1β stimulation in human articular chondrocytes. A specific STAT1 inhibitor, fludarabine, attenuates the hyper-expression of C3 and delays/prevents spontaneous OA in Dunkin-Hartley guinea pigs. Since fludarabine is already clinically used for chemotherapy, this study has great translational potential as a unique disease-modifying osteoarthritis drug (DMOAD) in treating primary OA.

Abstract 1705: 3D growth modulates the competition between STAT3 and STAT5 in breast cancer

Abstract Approximately 13% of women are diagnosed with invasive breast cancer. Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that is often inappropriately activated in breast cancer and is frequently associated with Triple Negative Breast Cancer (TNBC). STAT5 can also be activated and inappropriate activation of STAT5 alone or with STAT3 is generally correlated with a more favorable prognosis, a lower grade tumor, and a better response to therapies compared to inappropriate activation of STAT3 alone. Therefore, knowing the activation status of STAT5 and understanding how STAT5 activation can attenuate STAT3-driven breast cancers could help guide therapeutic choices and patient treatment. STAT3 and STAT5 can regulate a set of overlapping target genes. For example, STAT3 and STAT5 can regulate BCL6 expression, where STAT5 represses expression and STAT3 enhances it. Importantly, STAT5 outcompetes and preferentially binds in the presence of activated STAT3. This suggests that the overlapping target genes could play an important role in understanding how activation of STAT5 can lead to a more favorable breast cancer compared to activation of STAT3 alone. This previous work was conducted in a 2D cell culture model; however, 3D models can better mimic clinical settings and the use of 3D models can help bridge the gap. Therefore, understanding the relationship between STAT3 and STAT5 in 3D is important. SK-BR-3 cells (HER2+ cells with low basal STAT3 activity) and MDA-MB-231 cells (TNBC cells with constitutive STAT3 activity) are being compared between 2D cell culture and 3D media suspension. We have shown using chromatin immunoprecipitation that STAT3 DNA-binding is enhanced in 3D at certain binding sites, whereas STAT5 is not. STAT5 has reduced dominance and preferential binding over STAT3 for BCL6 in 3D compared to 2D. In other cases, STAT3 binding is decreased upon STAT5 activation in 3D but not 2D. To begin to understand these differences, we analyzed phosphorylation of these STATs in 2D and 3D. Phospho-STAT3 levels were similar between 2D and 3D in SK-BR-3 cells when activated with LIF stimulation but were higher in 3D in the MDA-MB-231 cells which contain constitutive STAT3 activity. STAT5 activation by prolactin stimulation did not directly affect STAT3 phosphorylation in 2D or 3D but does affect STAT3 binding in 3D growing cells. However, prolactin induced phospho-STAT5 seems to be reduced in 3D in both cell lines. We are currently analyzing additional binding sites to better define the differences between STAT3 and STAT5 mediated gene regulation in 2D and 3D since STAT phosphorylation does not fully address it. Overall, our data suggests that differences between 2D and 3D breast cancer models will be important for translation to patient tumors and understanding the relationship between STAT3 and STAT5 will help elucidate how STAT5 activation status can be used when treating patients with breast cancer. Citation Format: Alexandra Elizabeth Temple, Sarah R. Walker. 3D growth modulates the competition between STAT3 and STAT5 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1705.

Abstract 3157: The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis

Abstract Brain metastasis (BM) affects approximately 10% of cancer patients with metastatic disease, representing a major cause of morbidity and mortality. BM is a complex multi-step process involving various immune checkpoint proteins. Mitogen-activated protein kinase (MAPK), particularly extracellular signal-regulated kinases 1/2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3) are implicated in tumorigenesis and are critical upstream regulators of Programmed Death Ligand 1 (PD-L1) expression, a common immunotherapy target. Tumor suppressor p53 is shown to regulate STAT3 and ERK1/2 signaling, which are dysregulated in various malignancies. We hypothesize that p53 along with STAT3 and ERK1/2 play a critical role in the initiation and maintenance of BM. Twenty-six BM tumors with various primary sites of origin were used following IRB guidelines. pSTAT3Tyr705, pERK1/2Thr202/Tyr204, mutant p53 (p53mt), and PD-L1 expression was determined by immunohistochemistry. Gene expression analysis of BM was performed on a cDNA microarray. Human brain metastatic breast cancer cells (MDA-MB-231) were treated with STAT3 inhibitor (NSC74859) or MAPK inhibitor (U0126) in regular or astrocytic media to determine cell proliferation and migration by MTT assay and scratch-wound, respectively. ERK1/2 signaling pathway status was determined by Western blotting. Results demonstrated: 1) pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were expressed profusely at tumor margins, while p53mt was uniformly expressed throughout. 81% of BM expressed p53mt while 86% and 63% expressed pERK1/2Thr202/Tyr204 and pSTAT3Tyr705, respectively. Venn analysis displayed significant overlap between expression, with 62% overlap between p53mt and pERK1/2Thr202/Tyr204 and a 50% overlap between both pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 with p53mt. Expression of PD-L1 was seen in a majority of BM tumors. 2) Gene expression analysis demonstrated alterations in 32 STAT3- and MAPK-associated genes; these included upregulation of stathmin-1 and stathmin-like 3, which control microtubule dynamics in STAT3-related cell migration, and changes in cytokine-related genes, suggesting altered immune responses. 3) Cell proliferation showed augmented growth in astrocytic media (p≤0.05) and was significantly decreased following treatment with NSC74850 or U0126 (p≤0.01). While migration showed a trend towards significance in reduction following NSC74859 (p=0.08), migration stagnated following U0126 treatment in astrocytic media (p≤0.01). 4) pERK1/2Thr202/Tyr204 expression was reduced following U0126 or NSC74859 treatment. These findings suggest activation of STAT3 and ERK1/2 promote BM and suppression of these pathways may lead to a reduction in cell viability and motility. Therefore, this provides compelling evidence for the use of STAT3 and ERK1/2 along with p53 as potential targets for immunotherapy in brain metastatic disease. Citation Format: Sabrina L. Zeller, Eris Spirollari, John V. Wainwright, Simon J. Hanft, Chirag D. Gandhi, Meena Jhanwar-Uniyal. The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3157.

Abstract 6539: RNAi mediated silencing of STAT3 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors

Abstract Malignant tumors are often associated with an immunosuppressive tumor microenvironment (TME), rendering most of them resistant to standard-of-care immune checkpoint inhibitors (CPIs). Signal transducer and activator of transcription 3 (STAT3), a ubiquitously expressed transcription factor, has well-defined immunosuppressive functions in several leukocyte populations within the TME. Since the STAT3 protein has been challenging to target using conventional pharmaceutical modalities, we investigated the feasibility of applying systemically delivered RNA interference (RNAi) agents to silence its mRNA directly in tumor-associated immune cells. In preclinical murine tumor models, chemically stabilized acetylated small-interfering RNAs (siRNAs) selectively silenced Stat3 mRNA in multiple relevant cell types in TME and tumor-draining lymph nodes (TDLN), reduced STAT3 protein levels, and increased cytotoxic T-cell infiltration. In murine models of CPI-resistant tumors, RNAi-mediated Stat3 silencing resulted in tumor growth inhibition, which was further enhanced and resulted in tumor regressions in combination with CPIs. Our findings suggest that targeting STAT3 directly and reducing STAT3 protein in the suppressive immune cell populations in the TME and TDLN may overcome some of the current limitations of immunotherapy and be of therapeutic benefit especially for cancer patients who failed to respond to standard immunotherapy treatments alone. A human active STAT3 conjugate is currently in Phase 1 clinical trial for treatment of immunotherapy refractory cancer types (NCT06098651). Citation Format: Shanthi Ganesh, Minju Kim, Jenny Lee, Xudong Feng, Harini Sivagurunatha Krishnan, Rene Rijnbrand, Amber Beaudry, Jennifer A. Lockridge, Vinita Uttamsingh, John Hanrahan. RNAi mediated silencing of STAT3 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6539.

5-AZA Upregulates SOCS3 and PTPN6/SHP1, Inhibiting STAT3 and Potentiating the Effects of AG490 against Primary Effusion Lymphoma Cells.

Epigenetic modifications, including aberrant DNA methylation occurring at the promoters of oncogenes and oncosuppressor genes and histone modifications, can contribute to carcinogenesis. Aberrant methylation mediated by histone methylatransferases, alongside histones, can affect methylation of proteins involved in the regulation of pro-survival pathways such as JAK/STAT and contribute to their activation. In this study, we used DNA or histone demethylating agents, 5-Azacytidine (5-AZA) or DS-3201 (valemetostat), respectively, to treat primary effusion lymphoma (PEL) cells, alone or in combination with AG490, a Signal transducer and activator of transcription 3 (STAT3) inhibitor. Cell viability was investigated by trypan blue assay and FACS analysis. The molecular changes induced by 5-AZA and/or AG490 treatments were investigated by Western blot analysis, while cytokine release by PEL cells treated by these drugs was evaluated by Luminex. Statistical analyses were performed with Graphpad Prism® software (version 9) and analyzed by Student's t test or a nonparametric one-way ANOVA test. The results obtained in this study suggest that 5-AZA upregulated molecules that inhibit STAT3 tyrosine phosphorylation, namely Suppressor of Cytokine Signaling 3 (SOCS3) and tyrosine-protein phosphatase non-receptor type (PTPN) 6/Src homology region 2 domain-containing phosphatase-1 (SHP-1), reducing STAT3 activation and downregulating several STAT3 pro-survival targets in PEL cells. As this lymphoma is highly dependent on the constitutive activation of STAT3, 5-AZA impaired PEL cell survival, and when used in combination with AG490 JAK2/STAT3 inhibitor, it potentiated its cytotoxic effect. Differently from 5-AZA, the inhibition of the EZH1/2 histone methyltransferase by DS-3201, reported to contribute to STAT3 activation in other cancers, slightly affected STAT3 phosphorylation or survival in PEL cells, either alone or in combination with AG490. This study suggests that 5-AZA, by upregulating the expression level of SOCS3 and PTPN6/SHP1, reduced STAT3 activation and improved the outcome of treatment targeting this transcription factor in PEL cells.

MCL1 inhibition: a promising approach to augment the efficacy of sorafenib in NSCLC through ferroptosis induction

Ferroptosis, an iron-dependent form of regulated cell death, plays a crucial role in modulating the therapeutic response in non-small cell lung cancer (NSCLC) patients. Studies have identified the signal transducer and activator of transcription 3 (STAT3) and myeloid cell leukemia-1 (MCL1) as potential targets for sorafenib, which exhibits activities in inducing ferroptosis. However, the role of STAT3-MCL1 axis in sorafenib-induced ferroptosis in NSCLC is still unclear. This study provided evidence that ferroptosis is a critical driver of sorafenib-induced cell death in NSCLC, supported by the accumulation of lipid peroxidation products, indicative of oxidative stress-induced cell death. Additionally, both in vitro and in vivo experiments showed that ferroptosis contributed to a significant portion of the anti-cancer effects elicited by sorafenib in NSCLC. The noticeable accumulation of lipid peroxidation products in sorafenib-treated mice underscored the significance of ferroptosis as a contributing factor to the therapeutic response of sorafenib in NSCLC. Furthermore, we identified the involvement of the STAT3/MCL1 axis in sorafenib-induced antitumor activity in NSCLC. Mechanistically, sorafenib inhibited endogenous STAT3 activation and downregulated MCL1 protein expression, consequently unleashing the ferroptosis driver BECN1 from the BECN1-MCL1 complex. Conversely, there is an augmented association of BECN1 with the catalytic subunit of system Xc−, SLC7A11, whose activity to import cystine and alleviate lipid peroxidation is hindered upon its binding with BECN1. Notably, we found that MCL1 upregulation correlated with ferroptosis resistance in NSCLC upon sorafenib treatment. Our findings highlight the importance of sorafenib-triggered ferroptosis in NSCLC and offer a novel strategy to treat advanced NSCLC patients: by downregulating MCL1 and, in turn, predispose NSCLC cells to ferroptosis.

Enhanced STAT3/PIK3R1/mTOR signaling triggers tubular cell inflammation and apoptosis in septic-induced acute kidney injury: implications for therapeutic intervention.

Septic acute kidney injury (AKI) is a severe form of renal dysfunction associated with high morbidity and mortality rates. However, the pathophysiological mechanisms underlying septic AKI remain incompletely understood. Herein, we investigated the signaling pathways involved in septic AKI using the mouse models of lipopolysaccharide (LPS) treatment and cecal ligation and puncture (CLP). In these models, renal inflammation and tubular cell apoptosis were accompanied by the aberrant activation of the mechanistic target of rapamycin (mTOR) and the signal transducer and activator of transcription 3 (STAT3) signaling pathways. Pharmacological inhibition of either mTOR or STAT3 significantly improved renal function and reduced apoptosis and inflammation. Interestingly, inhibition of STAT3 with pharmacological inhibitors or small interfering RNA blocked LPS-induced mTOR activation in renal tubular cells, indicating a role of STAT3 in mTOR activation. Moreover, knockdown of STAT3 reduced the expression of the phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1/p85α), a key subunit of the phosphatidylinositol 3-kinase for AKT and mTOR activation. Chromatin immunoprecipitation assay also proved the binding of STAT3 to PIK3R1 gene promoter in LPS-treated kidney tubular cells. In addition, knockdown of PIK3R1 suppressed mTOR activation during LPS treatment. These findings highlight the dysregulation of mTOR and STAT3 pathways as critical mechanisms underlying the inflammatory and apoptotic phenotypes observed in renal tubular cells during septic AKI, suggesting the STAT3/ PIK3R1/mTOR pathway as a therapeutic target of septic AKI.

Toosendanin induces hepatotoxicity by restraining autophagy and lysosomal function through inhibiting STAT3/CTSC axis

Toosendanin (TSN) is the main active component in the traditional herb Melia toosendan Siebold & Zucc, which exhibits promising potential for development due to its diverse pharmacological properties. However, the hepatotoxicity associated with TSN needs further investigation. Previous research has implicated autophagy dysregulation in TSN-induced hepatotoxicity, yet the underlying mechanisms remain elusive. In this study, the mechanisms of signal transducer and activator of transcription 3 (STAT3) in TSN-induced autophagy inhibition and liver injury were explored using Stat3 knockout C57BL/6 mice and HepG2 cells. TSN decreased cell viability, increased lactate dehydrogenase (LDH) production in vitro, and elevated serum aspartate transaminase (AST) and alanine aminotransferase (ALT) levels as well as liver lesions in vivo, suggesting TSN had significant hepatotoxicity. TSN inhibited Janus kinase 2 (JAK2)/STAT3 pathway and the expression of cathepsin C (CTSC). Inhibition of STAT3 exacerbated TSN-induced autophagy inhibition and hepatic injury, whereas activation of STAT3 attenuated these effects of TSN. Mechanistically, STAT3 transcriptionally regulated the level of CTSC gene, which in turn affected autophagy and the process of liver injury. TSN-administered Stat3 knockout mice showed more severe hepatotoxicity, CTSC downregulation, and autophagy blockade than wildtype mice. In summary, TSN caused hepatotoxicity by inhibiting STAT3/CTSC axis-dependent autophagy and lysosomal function.

Fucoxanthin induces human melanoma cytotoxicity by thwarting the JAK2/STAT3/BCL-xL signaling axis.

Melanoma is the most lethal skin malignancy. Fucoxanthin is a marine carotenoid with significant anticancer activities. Intriguingly, Fucoxanthin's impact on human melanoma remains elusive. Signal Transducer and Activator of Transcription 3 (STAT3) represents a promising target in cancer therapy due to its persistent activation in various cancers, including melanoma. Herein, we revealed that Fucoxanthin is cytotoxic to human melanoma cell lines A2758 and A375 while showing limited cytotoxicity to normal human melanocytes. Apoptosis is a primary reason for Fucoxanthin's melanoma cytotoxicity, as the pan-caspase inhibitor z-VAD-fmk drastically abrogated Fucoxanthin-elicited clonogenicity blockage. Besides, Fucoxanthin downregulated tyrosine 705-phosphorylated STAT3 (p-STAT3 (Y705)), either inherently present in melanoma cells or inducible by interleukin 6 (IL-6) stimulation. Notably, ectopic expression of STAT3-C, a dominant-active STAT3 mutant, abolished Fucoxanthin-elicited melanoma cell apoptosis and clonogenicity inhibition, supporting the pivotal role of STAT3 blockage in Fucoxanthin's melanoma cytotoxicity. Moreover, Fucoxanthin lowered BCL-xL levels by blocking STAT3 activation, while ectopic BCL-xL expression rescued melanoma cells from Fucoxanthin-induced killing. Lastly, Fucoxanthin was found to diminish the levels of JAK2 with dual phosphorylation at tyrosine residues 1007 and 1008 in melanoma cells, suggesting that Fucoxanthin impairs STAT3 signaling by blocking JAK2 activation. Collectively, we present the first evidence that Fucoxanthin is cytotoxic selectively against human melanoma cells while sparing normal melanocytes. Mechanistically, Fucoxanthin targets the JAK2/STAT3/BCL-xL antiapoptotic axis to provoke melanoma cell death. This discovery implicates the potential application of Fucoxanthin as a chemopreventive or therapeutic strategy for melanoma management.

Anti-Inflammatory Effects of Chamaecyparis obtusa (Siebold & Zucc.) Endl. Leaf Extract Fermented by Ganoderma applanatum Mycelia.

Corticosteroids are commonly used anti-inflammatory agents. However, their prolonged use can lead to side effects. Therefore, the development of natural compounds with minimal side effects is necessary. This study was performed to investigate the anti-inflammatory effects and mechanisms of action of Chamaecyparis obtusa (Siebold & Zucc.) Endl. leaf (COL), bioconverted using Ganoderma applanatum (G. applanatum) in lipopolysaccharide (LPS)-induced RAW264.7 cells. The COL 70% EtOH extract fermented by G. applanatum (70COLGA) improved the high cytotoxicity of 70% EtOH extracts (70COL). When RAW264.7 cells were pre-treated with 100 and 200 μg/mL of 70COLGA for 2 h and then treated with LPS for 16 h, LPS induced the production of nitric oxide (NO), and the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) were significantly inhibited. When RAW264.7 cells were pre-treated with 100 and 200 μg/mL of 70COLGA for 2 h and then treated with LPS for 4 h, the phosphorylation of signal transducers and activators of transcription (STAT) was markedly decreased. In addition, 70COLGA markedly suppressed the production of the inflammatory cytokines interleukin (IL)-1β and IL-6 in LPS-induced RAW264.7 cells. Analysis of pro-inflammatory molecules using cytokine arrays showed that macrophage inflammatory protein (MIP)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and IL-27 expressions were also suppressed by 200 μg/mL of 70COLGA in LPS-induced RAW264.7 cells. These results demonstrate that 70COLGA significantly prevented inflammatory responses by inhibiting the secretion of pro-inflammatory molecules in LPS-induced RAW264.7 cells. When RAW264.7 cells were pre-treated with 100 and 200 μg/mL of 70COLGA for 2 h and then treated with LPS-conditioned medium (LPS-CM) for 30 min, 70COLGA directly inhibited STAT activation. In summary, our findings suggest that 70COLGA has therapeutic potential for the treatment of inflammatory diseases.

2'-Hydroxycinnamaldehyde Alleviates Intestinal Inflammation by Attenuating Intestinal Mucosal Barrier Damage Via Directly Inhibiting STAT3.

The currently available clinical therapeutic drugs for ulcerative colitis (UC) are considered inadequate owing to certain limitations. There have been reports on the anti-inflammatory effects of 2'-hydroxycinnamaldehyde (HCA). However, whether HCA can improve UC is still unclear. Here, we aimed to investigate the pharmacological effects of HCA on UC and its underlying molecular mechanisms. The pharmacological effects of HCA were comprehensively investigated in 2 experimental setups: mice with dextran sulfate sodium (DSS)-induced colitis and lipopolysaccharide (LPS)-treated fetal human colon (FHC) cells. Furthermore, the interaction between HCA and signal transducer and activator of transcription 3 (STAT3) was investigated using molecular docking. The FHC cells with STAT3 knockdown or overexpression and mice with intestinal epithelium-specific STAT3 deletion (STAT3ΔIEC) were used to evaluate whether STAT3 mediated the pharmacological effects of HCA. 2'-Hydroxycinnamaldehyde attenuated dysregulated expression of inflammatory cytokines in a dose-dependent manner while increasing the expression of tight junction proteins, reducing the apoptosis of intestinal epithelial cells, and effectively alleviating inflammation both in vivo and in vitro. 2'-Hydroxycinnamaldehyde bound directly to STAT3 and inhibited its activation. The modulation of STAT3 activation levels due to STAT3 knockdown or overexpression influenced the mitigating effects of HCA on colitis. Further analysis indicated that the remission effect of HCA was not observed in STAT3ΔIEC mice, indicating that STAT3 mediated the anti-inflammatory effects of HCA. We present a novel finding that HCA reduces colitis severity by attenuating intestinal mucosal barrier damage via STAT3. This discovery holds promise as a potential new strategy to alleviate UC.

Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment.

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. Unfortunately, targeting STAT3 with small molecules has proven to be very challenging, and for full activation of STAT3, the cooperative phosphorylation of both tyrosine 705 (Tyr705) and serine 727 (Ser727) is needed. Further, a selective inhibitor of STAT3 dual phosphorylation has not been developed. Here, we identified a low nanomolar potency and highly selective small-molecule STAT3 inhibitor that simultaneously inhibits both STAT3 Tyr705 and Ser727 phosphorylation. YY002 potently inhibited STAT3-dependent tumor cell growth in vitro and achieved potent suppression of tumor growth and metastasis in vivo. More importantly, YY002 exhibited favorable pharmacokinetics, an acceptable safety profile, and superior antitumor efficacy compared to BBI608 (STAT3 inhibitor that has advanced into phase III trials). For the mechanism, YY002 is selectively bound to the STAT3 Src Homology 2 (SH2) domain over other STAT members, which strongly suppressed STAT3 nuclear and mitochondrial functions in STAT3-dependent cells. Collectively, this study suggests the potential of small-molecule STAT3 inhibitors as possible anticancer therapeutic agents.

Molecular crosstalk between MUC1 and STAT3 influences the anti-proliferative effect of Napabucasin in epithelial cancers

MUC1 is a transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in epithelial cancers. The cytoplasmic tail of MUC1 (MUC1 CT) aids in tumorigenesis by upregulating the expression of multiple oncogenes. Signal transducer and activator of transcription 3 (STAT3) plays a crucial role in several cellular processes and is aberrantly activated in many cancers. In this study, we focus on recent evidence suggesting that STAT3 and MUC1 regulate each other’s expression in cancer cells in an auto-inductive loop and found that their interaction plays a prominent role in mediating epithelial-to-mesenchymal transition (EMT) and drug resistance. The STAT3 inhibitor Napabucasin was in clinical trials but was discontinued due to futility. We found that higher expression of MUC1 increased the sensitivity of cancer cells to Napabucasin. Therefore, high-MUC1 tumors may have a better outcome to Napabucasin therapy. We report how MUC1 regulates STAT3 activity and provide a new perspective on repurposing the STAT3-inhibitor Napabucasin to improve clinical outcome of epithelial cancer treatment.