Signal Transducer And Activator Of Transcription 3 Inhibitor Research Articles

STAT3 Pathways Contribute to β-HCH Interference with Anticancer Tyrosine Kinase Inhibitors.

Organochlorine pesticides (OCPs) are a class of environmentally persistent and bioaccumulative pollutants. Among these, β-hexachlorocyclohexane (β-HCH) is a byproduct of lindane synthesis, one of the most worldwide widespread pesticides. β-HCH cellular mechanisms inducing chemical carcinogenesis correspond to many of those inducing chemoresistance, in particular, by the activation of signal transducer and activator of transcription 3 (STAT3) signaling pathways. For this purpose, four cell lines, representative of breast, lung, prostate, and hepatocellular cancers, were treated with β-HCH, specific tyrosine kinase inhibitors (TKIs), and a STAT3 inhibitor. All cell samples were analyzed by a viability assay, immunoblotting analysis, a wound-healing assay, and a colony formation assay. The results show that β-HCH reduces the efficacy of TKIs. The STAT3 protein, in this context, plays a central role. In fact, by inhibiting its activity, the efficacy of the anticancer drug is restored. Furthermore, this manuscript aimed to draw the attention of the scientific and socio-healthcare community to the issue of prolonged exposure to contaminants and their impact on drug efficacy.

STAT3-induced lncRNA GNAS-AS1 accelerates keloid formation by mediating the miR-196a-5p/CXCL12/STAT3 axis in a feedback loop.

Keloids are pathological scar tissue resulting from skin trauma or spontaneous formation, often accompanied by itching and pain. Although GNAS antisense RNA 1 (GNAS-AS1) shows abnormal upregulation in keloids, the underlying molecular mechanism is unclear. The levels of genes and proteins in clinical tissues from patients with keloids and human keloid fibroblasts (HKFs) were measured using quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assay. The features of HKFs, including proliferation and migration, were evaluated using cell counting kit 8 and a wound healing assay. The colocalization of GNAS-AS1 and miR-196a-5p in HKFs was measured using fluorescence insitu hybridization. The relationships among GNAS-AS1, miR-196a-5p and C-X-C motif chemokine ligand 12 (CXCL12) in samples from patients with keloids were analysed by Pearson correlation analysis. Gene interactions were validated by chromatin immunoprecipitation and luciferase reporter assays. GNAS-AS1 and CXCL12 expression were upregulated and miR-196a-5p expression was downregulated in clinical tissues from patients with keloids. GNAS-AS1 knockdown inhibited proliferation, migration, and extracellular matrix (ECM) accumulation of HKFs, all of which were reversed by miR-196a-5p downregulation. Signal transducer and activator of transcription 3 (STAT3) induced GNAS-AS1 transcription through GNAS-AS1 promoter interaction, and niclosamide, a STAT3 inhibitor, decreased GNAS-AS1 expression. GNAS-AS1 positively regulated CXCL12 by sponging miR-196-5p. Furthermore, CXCL12 knockdown restrained STAT3 phosphorylation in HKFs. Our findings revealed a feedback loop of STAT3/GNAS-AS1/miR-196a-5p/CXCL12/STAT3 that promoted HKF proliferation, migration and ECM accumulation and affected keloid progression.

Uridine phosphorylase-1 promotes cell viability and cell-cycle progression in human epidermal keratinocytes via the glycolytic pathway.

Glycolysis is vital for the excessive proliferation of keratinocytes in psoriasis, and uridine phosphorylase-1 (UPP1) functions as an enhancer of cancer cell proliferation. However, little is known about whether UPP1 promotes keratinocyte proliferation and accelerates psoriasis development. This study revealed that UPP1 facilitates cell viability and cell-cycle progression in human epidermal keratinocytes (HEKs) by modulating the glycolytic pathway. Bioinformatics analysis of UPP1 gene expression and its correlation with the Reactome revealed that UPP1 mRNA expression, cell-cycle progression, the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway and glycolysis were positively associated with psoriasis. Cell proliferation, the cell cycle and glycolysis were evaluated after UPP1 was silenced or overexpressed. The results showed that UPP1 overexpression increased cell proliferation, cell-cycle progression and glycolysis, which was contrary to the effects of UPP1 silencing. However, the STAT3 inhibitor diminished UPP1 expression because STAT3 can bind to the UPP1 promoter. In conclusion, UPP1 was significantly activated by the IL-6/STAT3 pathway and could modulate glycolysis to regulate cell proliferation and cell-cycle progression in keratinocytes during the development of psoriasis.

#2949 Diverse effect of STAT3 inhibition on murine primary tubular epithelial cells

Abstract Background and Aims The incidence of chronic kidney diseases increases worldwide, and kidney fibrosis is the final common pathway regardless of the etiology. TGF-β plays a crucial role in the pathogenesis, affecting pro-fibrotic genes and transcription factors. STAT3 (signal transducer and activator of transcription 3) acts via the JAK/STAT signaling. Inhibition of STAT3 in animal studies was shown to attenuate the progression of kidney fibrosis. Yet, the effect of STAT3 inhibition on renal tubular epithelial cells has not been elucidated. We aimed to investigate in vitro the impact of pharmacological STAT3 inhibition with NSC74859 (NSC) and STATTIC on the expression of fibrosis associated genes of primary tubular epithelial cells. Method Primary tubular epithelial cells from kidneys of C57Bl6/J mice were isolated and used in our studies. Cells (P10-P15) were seeded on gelatin-coated 6-well culture plates (100000 cells/well) and subjected to treatment with 10 ng/ml TGF-β, 100 µM NSC, 10 µM STATTIC or vehicle (n = 3/group) as follows: Control; NSC; STATTIC; TGF-β; TGF-β+NSC; TGF-β+STATTIC. After 24 h treatment, mRNA and protein expressions were analyzed. Statistical evaluation was performed using Kruskal-Wallis test. Results In cells not treated with TGF-β, neither NSC nor STATTIC treatment affected the expression of Tgfb1 (TGF-β) and Acta2 (SMA) genes compared to controls (100%), but reduced Col1a1 expression to 58% and 54%, respectively (p < 0.05). NSC reduced to 54%, while STATTIC tended to increase (169%) the gene expression of Egr2 transcription factor. TGF-β treatment induced STAT3 phosphorylation by 45% accompanied by a fibrotic response and epithelial-to-mesenchymal transition (EMT) as indicated by significantly increased Tgfb1, Acta2, Egr2 and Col1a1 (p < 0.05). NSC treatment did not inhibit the TGF-β-induced Tgfb1 upregulation (178%), but markedly, although not significantly, reduced the expression of Acta2 (100%), Egr2 (98%) and Col1a1 (114%) in the TGF-β+NSC group. Interestingly, although STATTIC treatment dramatically inhibited STAT3 expression (38%, p < 0.05) it significantly enhanced the TGF-β-induced overexpression of Tgfb1 (210%, p < 0.01) and Egr2 (307%, p < 0.05), while markedly reduced Acta2 (64%, p < 0.001) and Col1a1 (81%, p < 0.01) in the TGF-β+STATTIC group. Conclusion Our results indicate that in vitro inhibition of STAT3 using two inhibitors with same mode of action lead to diverse responses in primary murine tubular epithelial cells, partially suppressing EMT markers but inducing pro-fibrotic EGR2. Further studies are needed to clarify the underlying mechanisms.

The STAT3 inhibitor B9 alleviates lipopolysaccharide-induced acute lung injury through its anti-inflammatory effects

The development of acute lung injury (ALI), a common respiratory condition with multiple causes, is significantly influenced by the pro-inflammatory environment of signal transducer and activator of transcription 3 (STAT3) in macrophages. Our study aimed to evaluate the anti-inflammatory effects of B9 (N-(4-hydroxyphenyl)-9, 10-dioxo-9, 10-dihydroanthracene-2-sulfonamide), a novel inhibitor targeting the STAT3 SH2 domain, in macrophages and to assess its therapeutic potential for ALI using a mouse model of lipopolysaccharide (LPS)-induced ALI. We found that B9 (30 mg/kg) significantly reduced lung pathological damage and neutrophil infiltration caused by the intratracheal administration of LPS. Additionally, the high expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in alveolar lavage fluid was also inhibited by B9 treatment. The decreased expression of CD86 and increased CD206 in lung tissue demonstrated the anti-inflammatory effect of B9, which was due to its inhibition of the STAT3 signaling pathway in macrophages of ALI mice. Furthermore, B9 suppressed the activation of RAW264.7 cells induced by LPS, characterized by its ability to inhibit the activation of iNOS and STAT3 in a dose-dependent manner, as well as reduce the secretion of IL-6 and IL-1β. The in vivo preliminary safety evaluation indicated that B9 had a favorable safety profile at the administered doses. These results suggest that B9 exerts a therapeutic effect on LPS-induced ALI, potentially by preventing the phosphorylation of STAT3 Y705 and S727 without affecting the STAT3 protein level. Taken together, these findings provide a foundation for developing B9 as a novel anti-inflammatory agent for ameliorating LPS-induced ALI.

Leptin Promotes Vasculogenic Mimicry in Breast Cancer Cells by Regulating Aquaporin-1.

Leptin is an obesity-related hormone that plays an important role in breast cancer progression. Vasculogenic mimicry (VM) refers to the formation of vascular channels lined by tumor cells. This study aimed to investigate the relationship between leptin and VM in human breast cancer cells. VM was measured by a 3D culture assay. Signal transducers and activators of transcription 3 (STAT3) signaling, aquaporin-1 (AQP1), and the expression of VM-related proteins, including vascular endothelial cadherin (VE-cadherin), twist, matrix metalloproteinase-2 (MMP-2), and laminin subunit 5 gamma-2 (LAMC2), were examined by Western blot. AQP1 mRNA was analyzed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Leptin increased VM and upregulated phospho-STAT3, VE-cadherin, twist, MMP-2, and LAMC2. These effects were inhibited by the leptin receptor-blocking peptide, Ob-R BP, and the STAT3 inhibitor, AG490. A positive correlation between leptin and AQP1 mRNA was observed and was confirmed by RT-PCR. Leptin upregulated AQP1 expression, which was blocked by Ob-R BP and AG490. AQP1 overexpression increased VM and the expression of VM-related proteins. AQP1 silencing inhibited leptin-induced VM and the expression of VM-related proteins. Thus, these results showed that leptin facilitates VM in breast cancer cells via the Ob-R/STAT3 pathway and that AQP1 is a key mediator in leptin-induced VM.

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.

Banxia Xiexin Decoction inhibits colitis-associated colorectal cancer development by modulating STAT3 signaling and gut microbiota

ObjectiveTo investigate the therapeutic effects of Banxia Xiexin Decoction (BXD), a herbal medicine formula, on inflammation and the imbalance of the gut microbiota in a rat model of colorectal cancer (CRC) induced by azoxymethane (AOM) /dextran sulfate sodium (DSS). MethodsA total of 75 male C57BL/6 mice were randomly divided into five groups: normal control group (NC), model group (MODEL), low-dose BXD treatment group (L-BXD), high-dose BXD treatment (H-BXD) group and MS treatment group (MS). BXD and MS were used in CRC mice at the doses of 3.915 g/kg, 15.66 g/kg, 0.6 g/kg for 3 weeks consecutively.Histopathological changes in the colon were observed using hematoxylin-eosin (HE) staining. The content of inflammatory factors in serum was detected by an enzyme-linked immunosorbent assay (ELISA), and the expression of mRNA and protein of genes related to immunity, apoptosis, inflammation, and inflammatory factors was evaluated. Changes in the intestinal flora of mouse fecal were determined based on high-throughput sequencing of the 16S rRNA microbial gene. ResultsCompared to the model group, the low-dose BXD and high-dose BXD groups decreased the number of colon tumors, reversed weight loss, and shortened colon length of mice. The pathological examination showed that BXD alleviated the malignancy of intestinal tumors. It also suppressed signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase-9 (MMP-9), and transforming growth factor beta 1 (TGF-β1) expression, while increasing the expression of the tight junction protein ZO-1 in colon tissues. Additionally, the levels of key pathway proteins involved in inflammation (phosphorylated-STAT3, Bcl-2, COX-2) and cell cycle regulatory molecules (c-Myc and PCNA) were reduced. According to 16S rRNA sequence analysis, BXD enhanced the relative abundance of potentially beneficial bacteria, while that of cancer-related bacteria decreased. ConclusionBXD plays a preventive role in developing colorectal cancer; its mechanisms are related to the inhibition of inflammation and tumor proliferation, as well as maintenance of intestinal homeostasis.

Inhibition of STAT3 by S3I-201 suppress peritoneal fibroblast phenotype conversion and alleviate peritoneal fibrosis.

Peritoneal fibrosis is a common pathological response to long-term peritoneal dialysis (PD) and a major cause for PD discontinuation. Understanding the cellular and molecular mechanisms underlying the induction and progression of peritoneal fibrosis is of great interest. In our study, invitro study revealed that signal transducer and activator of transcription 3 (STAT3) is a key factor in fibroblast activation and extracellular matrix (ECM) synthesis. Furthermore, STAT3 induced by IL-6 trans-signalling pathway mediate the fibroblasts of the peritoneal stroma contributed to peritoneal fibrosis. Inhibition of STAT3 exerts an antifibrotic effect by attenuating fibroblast activation and ECM production with an invitro co-culture model. Moreover, STAT3 plays an important role in the peritoneal fibrosis in an animal model of peritoneal fibrosis developed in mice. Blocking STAT3 can reduce the peritoneal morphological changes induced by chlorhexidine gluconate. In conclusion, our findings suggested STAT3 signalling played an important role in peritoneal fibrosis. Therefore, blocking STAT3 might become a potential treatment strategy in peritoneal fibrosis.

Identification of Novel STAT3 Dimerization Inhibitor Through Structure-Based Virtual Screening for Cancer Management

Background: Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that controls cell proliferation, differentiation, angiogenesis, and immunological responses. In many human malignancies, abnormal STAT3 activation promotes tumor growth via oncogenic gene expression, resulting in tumor malignancy. Many drugs with clinically authorized analogues that are used as STAT3 inhibitors for cancer therapy have several drawbacks in terms of stability and toxicity.Methods: This study used PyRx 0.8 tool to screen the Traditional Chinese Medicine (TCM) database of about 32,364 commercially available natural compounds in order to identify new STAT3 inhibitors. Physicochemical and ADME properties of selected compounds were estimated using Datawarrior and SwissADME.Result: The top 20 compounds were initially chosen based on their strong binding affinities with STAT3. Lipinski and Vaber tools were used to filter the top 20 compounds, yielding the top 6 compounds. The compounds ZINC85542844, ZINC4098720, ZINC85543599, ZINC85593523, ZINC85593528, and ZINC85593537 were passed through these filters. These compounds were found to interact with active site STAT3 residues and have several amino acid interactions in common with the control compound (STX0119).Conclusion: This study suggests that the compounds ZINC85542844, ZINC4098720, ZINC85543599, ZINC85593523, ZINC85593528, and ZINC85593537 could be used as a lead for the development of novel STAT3 inhibitors. However, further experimental validation is required to optimized them as STAT3 inhibitors.Keywords: STAT3; Transcription factor; Natural compounds; Cancer; Virtual screening

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.

STAT3 promotes cytoplasmic-nuclear translocation of RNA-binding protein HuR to inhibit IL-1β-induced IL-8 production

Signal transducer and activator of transcription 3 (STAT3) functions to regulate inflammation and immune response, but its mechanism is not fully understood. We report here that STAT3 inhibitors Stattic and Niclosamide up-regulated IL-1β-induced IL-8 production in C33A, CaSki, and Siha cervical cancer cells. As expected, IL-1β-induced IL-8 production was also up-regulated through the molecular inhibition of STAT3 by use of CRISPR/Cas9 technology. Unexpectedly, IL-1β induced IL-8 production via activating ERK and P38 signal pathways, but neither STAT3 inhibitors nor STAT3 knockout affected IL-1β-induced signal transduction, suggesting that STAT3 decreases IL-8 production not via inhibition of signal transduction. To our surprise, STAT3 inhibition increased the stabilization, and decreased the degradation of IL-8 mRNA, suggesting a post-transcriptional regulation of IL-1β-induced IL-8. Moreover, Dihydrotanshinone I, an inhibitor of RNA-binding protein HuR, down-regulated IL-1β-induced IL-8 dose-dependently. HuR inhibition by CRISPR/Cas9 also decreased IL-8 production induced by IL-1β. Mechanistically, co-immunoprecipitation results showed that STAT3 did not react with HuR directly, but STAT3 inhibition increased the protein levels of HuR in cytoplasm. And IL-6 activation of STAT3 induced HuR cytoplasmic-nuclear transport. Taken together, these results suggest that STAT3 contributes to HuR nuclear localization and inhibits Il-1β-induced IL-8 production through this non-transcriptional mechanism.

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 3049: Similarities between ovarian cancer and endometriosis: The role of STAT3

Abstract Endometriosis is a painful gynecological disease that affects over 190 million women worldwide. This disease is classified by the presence of endometrial-like cells growing in and around the female reproductive system. Instead of lining the uterus, these ectopic cells shed off and cluster together forming lesions that attach to different surfaces of the female reproductive system. In particular, endometriosis cells interact with and form lesions within the mesothelial cells that line the peritoneum. There is currently no cure, and many of the therapies aiming to help with pain management come with unfavorable tradeoffs. Our research on ovarian cancer led us to investigate potential overlapping characteristics with endometriosis. Growing ovarian cancer cells in 3D causes spheroids to form which mimic the physical features of tumors and are also able to better represent cell-cell and cell-environment interactions. Analysis of the endometriosis cell lines 12Z and hEM3 demonstrated that they could also form spheroids. As both ovarian cancer cells and endometriosis cells interact with mesothelial cells, we wanted to determine if endometriosis spheroids were able to clear the mesothelial layer comparable to ovarian cancer spheroids. Evaluation of 12Z and hEM3 cell lines demonstrated that endometriosis spheroids were able to undergo mesothelial clearance. While the exact cause of endometriosis is not known, different proteins have been identified as important factors in the progression of endometriosis. The Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor involved in cell differentiation, proliferation, apoptosis resistance, angiogenesis, and metastasis. In endometriosis cell lines, STAT3 is constitutively active, which makes it a promising potential therapeutic target. To verify that STAT3 is important in endometriosis, siRNA was used to reduce the expression of STAT3 in two endometriosis cell lines, 12Z and hEM3. Both cell lines showed a decrease in cell viability. In addition, reducing the expression of STAT3 in the endometriosis cells appeared to decrease mesothelial clearance ability. Our lab previously identified statins as potential STAT3 inhibitors by using the Broad Institute’s CLUE tool. After statin treatment, 12Z and hEM3 spheroids showed a decrease in cell viability and clearance ability. Taken together, these data suggest that STAT3 plays a key role in endometriosis and that the repurposing of statins may provide a benefit to patients with endometriosis. Citation Format: Allison A. Kloeckner, Sarah R. Walker. Similarities between ovarian cancer and endometriosis: The role of STAT3 [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 3049.

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.

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.

Abstract WP301: STAT3 Inhibition Reduces Infarct and Neuroinflammation in Experimental Stroke

Signal Transducer and Activator of Transcription 3 (STAT3) activation is increased in the brain following cerebral ischemia. STAT3, a transcription factor, is a positive regulator of cell survival and is implicated in cytoprotection following ischemia in multiple cell types, including neurons and endothelial cells. We hypothesized that pharmacological inhibition of STAT3 activation in mice during the reperfusion phase after middle cerebral artery occlusion (MCAO) will lead to increased infarct size. We subjected mice to 1-hour MCAO, following which they received STAT3 inhibitor Stattic (20 mg/kg), or vehicle, for 24 hours delivered subcutaneously via osmotic pump. Twenty-four hours later, infarct was measured by TTC and inflammatory cells were measured by flow cytometry. For experiments involving splenectomy, spleens were surgically removed 14 days prior to MCAO surgery. Surprisingly, we found that STAT3 inhibition reduces infarct after MCAO, with infarct in the hemisphere reducing from 49.90 ± 2.44 % in vehicle to 32.86 ± 1.55 % in Stattic-treated mice. Since STAT3 is involved in inflammatory processes, as well as cytoprotection, this led us to hypothesize that the reduction in infarct we observe may be due to decreased inflammation. We carried out FACS analysis and observed that Stattic inhibits MCAO-induced infiltration of B-, T- and dendritic cells into the brain. Neutrophil and monocyte infiltration into the brain were unaffected by Stattic. Since MCAO is known to induce splenic atrophy, with spleen size being inversely proportional to infarct volume, we measured spleen weights. Interestingly Stattic-treated mice had larger spleens (66.31 ± 5.40 g) compared to vehicle-treated mice (46.43 ± 4.60 g), indicating a dampened peripheral immune response upon inhibition of STAT3. Furthermore, Stattic was no longer effective in reducing infarct size in splenectomized mice. We conclude that pharmacological inhibition of STAT3 reduces infarct following MCAO, with the mechanism of protection involving differential regulation of the peripheral immune response.

Abstract B037: Utilizing GSK3β and integrin inhibitors to target STAT3 activity in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer known for its high rate of recurrence and metastatic ability. Patients face a poor prognosis and the five-year survival rate for those diagnosed with metastatic TNBC is very low. The transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) is constitutively active in TNBC and functions to promote proliferation and metastasis. Therefore, it is a potential therapeutic target for TNBC. We used CLUE, an online bioinformatics tool from the Broad Institute, to identify potential STAT3 inhibitors. To do this, a STAT3 gene signature generated from TNBC cells was used to query the gene signatures from drug treatments in the CLUE dataset for compounds that caused a similar signature. The hypothesis is that a drug with a similar signature to the STAT3 inhibition signature would be a potential STAT3 inhibitor. Drugs were chosen based on high similarity rating and known function. One of the top drugs we chose to pursue was TWS-119, a GSK3β inhibitor. GSK3β is a serine/threonine kinase that regulates proliferation and cell survival. TWS-119 was found to decrease the viability of MDA-MB-231, MDA-MB-468, and Hs587T TNBC cell lines. To assess the ability of TWS-119 to inhibit STAT3 activity, we used a STAT3 responsive reporter and found that TWS-119 inhibited STAT3 activity in MDA-MB-468 cells. To determine the mechanism of inhibition of STAT3 activity, we analyzed the effects of TWS-119 on STAT3 tyrosine phosphorylation. Preliminary results suggest that TWS-119 inhibits STAT3 activity via inhibition of tyrosine phosphorylation. Having determined that TWS-119 inhibited STAT3 activity, we wanted to determine if TWS-119 had any effect on TNBC migration. We found that TWS-119 reduced the migratory ability of MDA-MB-231 cells. An additional drug identified from CLUE was Cilengitide, an inhibitor of integrin ⍺vβ3 which plays an important role in proliferation and migration. While Cilengitide only slightly decreased the viability of the three different TNBC cell lines (MDA-MB-231, MDA-MB-468, Hs587T), we found that combinations of Cilengitide and TWS-119 had synergistic effects on cell viability in the TNBC cells. Preliminary western blot analysis demonstrated that the combination of both drugs reduced the level of pY-STAT3. This suggests that this combination targeting STAT3 may be a viable treatment strategy for TNBC. However, the mechanism through which these drugs interact is unknown, and the cell signaling relationship between their known targets (GSK3β and integrin ⍺vβ3) and STAT3 is unclear. We are currently working to define the mechanism of action of these combination effects in TNBC cells. Using TWS-119 and Cilengitide, we can determine the connection between integrins, GSK3β, and STAT3 to better understand proliferation and metastasis and to identify new therapeutic options for TNBC patients. Citation Format: Emily A Pratt, Sarah R Walker. Utilizing GSK3β and integrin inhibitors to target STAT3 activity in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B037.