STAT3 Tyrosine Research Articles

Aiolos promotes CXCR3 expression on TH1 cells via positive regulation of IFNγ/STAT1 signaling.

CD4+ T helper 1 (TH1) cells coordinate adaptive immune responses to intracellular pathogens, including viruses. Key to this function is the ability of TH1 cells to migrate within secondary lymphoid tissues, as well as to sites of inflammation, which relies on signals received through the chemokine receptor CXCR3. CXCR3 expression is driven by the TH1 lineage-defining transcription factor T-bet, and the cytokine-responsive Signal Transducer and Activator of Transcription (STAT) family members STAT1 and STAT4. Here, we identify the Ikaros zinc finger (IkZF) transcription factor Aiolos (Ikzf3) as an additional positive regulator of CXCR3 both in vitro and in vivo using a murine model of influenza virus infection. Mechanistically, we find that Aiolos-deficient CD4+ T cells exhibit decreased expression of key components of the IFNγ/STAT1 signaling pathway, including JAK2 and STAT1. Consequently, Aiolos deficiency results in decreased levels of STAT1 tyrosine phosphorylation and reduced STAT1 enrichment at the Cxcr3 promoter. We further find that Aiolos and STAT1 form a positive feedback loop via reciprocal regulation of each other downstream of IFNγ signaling. Collectively, our study demonstrates that Aiolos promotes CXCR3 expression on TH1 cells by propagating the IFNγ/STAT1 cytokine signaling pathway.

Abstract 663: Irreversible covalent azetidine-based small molecule inhibitors of Stat3 activity block growth of human pancreatic tumors

Abstract The incidence and death rates for pancreatic adenocarcinoma in the US have been increasing by about 1% per year and the death rate has increased by approximately 0.2% yearly. The five-year survival rate has remained below 10% for many years. With surgery, radiation, and chemotherapy being the main treatment options to extend survival or for symptom relief, there is an urgent unmet need for effective new treatment options. Signal Transducer and Activator of Transcription (Stat) 3, one of the Stat family members, is aberrantly activated in human pancreatic and many other cancers. Aberrantly-active Stat3 promotes abnormal tumor-cell intrinsic and extrinsic mechanisms, including dysregulation of gene expression and mitochondria energy metabolism in the tumor cells, and suppression of immune cell functions in the tumor microenvironment. Stat3 is therefore an important target for therapeutic development. Herein we present two small molecules, H182 and H333, which are covalent inhibitors of Stat3 and that potently block Stat3 DNA-binding activity in vitro, with IC50 0.66-0.98 µM. H182 and its structural analog, H333 bind Stat3 via irreversible covalent interactions with key cysteine residues in the Stat3 DNA-binding domain to induce a time-dependent inhibition of Stat3 activity. Both compounds inhibit intracellular constitutive Stat3 tyrosine phosphorylation and induce loss of viable cells and apoptosis in vitro of human pancreatic Panc-1 and Mia-Paca2 cells. H333 is 13-fold more specific and H182 is 4-fold more specific against tumor cells over normal cells. Furthermore, MiaPaca-2 cells treated with H182 and analyzed by Seahorse showed dramatically decreased mitochondria respiration. Notably, xenograft models of MiaPaca2 treated with H182 or H333 via i.p. every other day for 27 days showed dramatic growth inhibition. Collectively, our results identify H182 and H333 as therapeutically-viable small molecules with unique irreversible mechanism of Stat3 inhibition which accounts for their strong antitumor effects against human pancreatic tumor xenografts. Citation Format: Yue Chen, Ning Zhai, Peibin Yue, Christine Brotherton-Pleiss, Wenzhen Fu, Kayo Nakamura, Weiliang Chen, Marcus Tius, Francisco Lopez-Tapia, James Turkson. Irreversible covalent azetidine-based small molecule inhibitors of Stat3 activity block growth of human pancreatic 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 663.

Upregulation of tumor suppressor PIAS3 by Honokiol promotes tumor cell apoptosis via selective inhibition of STAT3 tyrosine 705 phosphorylation.

The natural product Honokiol exhibits robust antitumor activity against a range of cancers, and it has also received approval to undergo phase I clinical trial testing. We confrmed that honokiol can promote the apoptotic death of tumor cells through cell experiments. Then siRNA constructs specific for PIAS3, PIAS3 overexpression plasmid and the mutation of the STAT3 Tyr705 residue were used to confirm the mechanism of Honokiol-induced apoptosis. Finally, we confrmed that honokiol can promote PIAS3 upregulation, in turn suppressing STAT3 Tyr705 phosphorylation through the in vivo and in vitro experiments. Honokiol was ultimately found to reduce tumor cell viability by promoting apoptosis through a mechanism dependent on the ability of Honokiol to promote PIAS3 upregulation and the selective inhibition of p-STAT3 (Tyr705) without affecting p-STAT3 (Ser727) or p-STAT1 (Tyr701) levels. PIAS3 knockdown and overexpression in tumor cells altered STAT3 activation and associated DNA binding activity through the control of Tyr705 phosphorylation via PIAS3-STAT3 complex formation, ultimately shaping Honokiol-induced tumor cell apoptosis. Honokiol was also confirmed to significantly prolong the survival of mice bearing xenograft tumors in a PIAS3-dependent fashion. Together, these findings highlight a novel pathway through which Honokiol can promote PIAS3 upregulation, in turn suppressing STAT3 Tyr705 phosphorylation and promoting the apoptotic death of tumor cells.

The mitochondrial succinate dehydrogenase complex controls the STAT3-IL-10 pathway in inflammatory macrophages

The functions of macrophages are tightly regulated by their metabolic state. However, the role of the mitochondrial electron transport chain (ETC) in macrophage functions remains understudied. Here, we provide evidence that the succinate dehydrogenase (SDH)/complex II (CII) is required for respiration and plays a role in controlling effector responses in macrophages. We find that the absence of the catalytic subunits Sdha and Sdhb in macrophages impairs their ability to effectively stabilize HIF-1α and produce the pro-inflammatory cytokine IL-1β in response to LPS stimulation. We also arrive at the novel result that both subunits are essential for the LPS-driven production of IL-10, a potent negative feedback regulator of the macrophage inflammatory response. This phenomenon is explained by the fact that the absence of Sdha and Sdhb leads to the inhibition of Stat3 tyrosine phosphorylation, caused partially by the excessive accumulation of mitochondrial reactive oxygen species (mitoROS) in the knockout cells.

Lachnochromonin, a fungal metabolite from Lachnum virgineum, inhibits cell growth and promotes apoptosis in tumor cells through JAK/STAT3 signaling

Natural compounds that interfere with tumor cell growth have potential to be used as therapeutic agents to treat cancers. Lachnochromonin (p71) is a small molecule isolated from Lachnum virgineum. Here, we reported the effect of p71 on human tumor cells, especially on breast cancer MCF-7 cells. We found that p71 significantly suppresses cell growth and induces apoptosis. The luciferase results demonstrated that p71 specifically attenuates the activation of JAK/STAT3 signaling. Biochemical analysis revealed that p71 blocks the phosphorylation of STAT3 tyrosine 705 and serine 727, resulting in down-regulation of c-Myc and Cyclin D1 expression level. Importantly, p71 inhibited cell growth, colony-formation, and migration through affecting STAT3 activity. These results implied that p71 may be used as a therapeutic agent against breast cancer.

445 Does tyrosine and serine phosphorylation of STAT3 drive cutaneous squamous cell carcinoma in recessive dystrophic epidermolysis bullosa?

The early onset of aggressive cutaneous squamous cell carcinoma (cSCC) and its rapid progression in recessive dystrophic epidermolysis bullosa (RDEB) leads to premature mortality. Elevated levels of STAT3Tyr705 (pY-STAT3) phosphorylation have been demonstrated in RDEB-derived cSCC cells, showing that constitutive activation and dysregulation of the pathway play a role in RDEB-cSCC pathogenesis. Application of Ruxolitinib (JAK1/2 inhibitor) to an RDEB-cSCC xenograph mouse model, reduces the size of small tumours but is insufficient to completely reduce bigger tumours.

Stat3 Tyrosine 705 and Serine 727 Phosphorylation Associate With Clinicopathological Characteristics and Distinct Tumor Cell Phenotypes in Triple-Negative Breast Cancer.

Signal transducer and activator of transcription 3 (Stat3) is responsible for many aspects of normal development and contributes to the development and progression of cancer through regulating epithelial cell identity and cancer stem cells. In breast cancer, Stat3 is associated with triple-negative breast cancers (TNBC) and its function has been related to the activation of p63, itself a marker of basal-like TNBC and a master regulator of stem cell activities. Stat3 activation is controlled by dual phosphorylation at tyrosine 705 (pTyr705) and serine 727 (pSer727), although it is unclear whether these have equivalent effects, and whether they are related or independent events. To address these issues, we investigated Stat3 phosphorylation at the two sites by immunohistochemistry in 173 patients with TNBC. Stat3 phosphorylation was assessed by automated quantitative measurements of digitized scanned images and classified into four categories based on histoscore. The results were analyzed for associations with multiple markers of tumor phenotype, proliferation, BRCA status, and clinicopathological characteristics. We show that the levels of pTyr705- and pSer727-Stat3 were independent in 34% of tumors. High pTyr705-Stat3 levels were associated with the luminal differentiation markers ERβ/AR and MUC1, whereas tumors with high levels of pSer727-Stat3 were more likely to be positive for the basal marker CK5/6, but were independent of p63 and were EGFR negative. Combined high pSer727- and low Tyr705-Stat3 phosphorylation associated with basal-like cancer. Although high Stat3 phosphorylation levels were associated with less aggressive tumor characteristics, they did not associate with improved survival, indicating that Stat3 phosphorylation is an unfavorable indicator for tumors with an otherwise good prognosis according to clinicopathological characteristics. These findings also show that pTyr705-Stat3 and pSer727-Stat3 associate with specific breast tumor phenotypes, implying that they exert distinct functional activities in breast cancer.

SS-4 is a highly selective small molecule inhibitor of STAT3 tyrosine phosphorylation that potently inhibits GBM tumorigenesis in vitro and in vivo

Glioblastoma (GBM) is a highly aggressive cancer with a dismal prognosis. Constitutively active STAT3 has a causal role in GBM progression and is associated with poor patient survival. We rationally designed a novel small molecule, SS-4, by computational modeling to specifically interact with STAT3. SS-4 strongly and selectively inhibited STAT3 tyrosine (Y)-705 phosphorylation in MT330 and LN229 GBM cells and inhibited their proliferation and induced apoptosis with an IC50 of ∼100 nM. The antiproliferative and apoptotic actions of SS-4 were Y-705 phosphorylation dependent, as evidenced by its lack of effects on STAT3 knockout (STAT3KO) cells or STAT3KO cells that overexpressed a phospho-Y705 deficient (STAT3Y705F) mutant, and the recovery of effects when wild-type STAT3 or a phospho-serine (S)727 deficient mutant was expressed in STAT3KO cells. SS-4 increased the expression of STAT3 repressed genes, while decreasing the expression of STAT3 promoted genes. Importantly, SS-4 markedly reduced the growth of GBM intracranial tumor xenografts. These data together identify SS-4 as a potent STAT3 inhibitor that selectively blocks Y705-phosphorylation, induces apoptosis, and inhibits growth of human GBM models in vitro and in vivo.

Hemgn Protects Hematopoietic Stem and Progenitor Cells Against Transplantation Stress Through Negatively Regulating IFN‐γ Signaling (Adv. Sci. 5/2022)

Hematopoietic Stem and Progenitor Cells In article number 2103838, Xiao-Ming Yang, Chang-Yan Li and co-workers identify a critical regulator-HEMGN, that is required for hematopoietic stem and progenitor cells (HSPCs) homing, survival, and reconstitution of blood cells during bone marrow transplantation (BMT). HEMGN acts as a protector which limits interferon-γ (IFN-γ) signaling by suppressing nuclear Stat1 tyrosine phosphorylation and assists HSPCs to combat the transplantation stress. Targeted Hemgn may be used to improve conditioning regimens and engraftment during bone marrow transplantation.

Hemgn Protects Hematopoietic Stem and Progenitor Cells Against Transplantation Stress Through Negatively Regulating IFN-γ Signaling.

Hematopoietic stem and progenitor cells (HSPCs) possess the remarkable ability to regenerate the whole blood system in response to ablated stress demands. Delineating the mechanisms that maintain HSPCs during regenerative stresses is increasingly important. Here, it is shown that Hemgn is significantly induced by hematopoietic stresses including irradiation and bone marrow transplantation (BMT). Hemgn deficiency does not disturb steady‐state hematopoiesis in young mice. Hemgn −/‐ HSPCs display defective engraftment activity during BMT with reduced homing and survival and increased apoptosis. Transcriptome profiling analysis reveals that upregulated genes in transplanted Hemgn −/− HSPCs are enriched for gene sets related to interferon gamma (IFN‐γ) signaling. Hemgn −/− HSPCs show enhanced responses to IFN‐γ treatment and increased aging over time. Blocking IFN‐γ signaling in irradiated recipients either pharmacologically or genetically rescues Hemgn −/‐ HSPCs engraftment defect. Mechanistical studies reveal that Hemgn deficiency sustain nuclear Stat1 tyrosine phosphorylation via suppressing T‐cell protein tyrosine phosphatase TC45 activity. Spermidine, a selective activator of TC45, rescues exacerbated phenotype of HSPCs in IFN‐γ‐treated Hemgn −/− mice. Collectively, these results identify that Hemgn is a critical regulator for successful engraftment and reconstitution of HSPCs in mice through negatively regulating IFN‐γ signaling. Targeted Hemgn may be used to improve conditioning regimens and engraftment during HSPCs transplantation.

Opposing Effects of Chelidonine on Tyrosine and Serine Phosphorylation of STAT3 in Human Uveal Melanoma Cells.

STAT3 is a transcription factor that regulates various cellular processes with oncogenic potential, thereby promoting tumorigenesis when activated uncontrolled. STAT3 activation is mediated by its tyrosine phosphorylation, triggering dimerization and nuclear translocation. STAT3 also contains a serine phosphorylation site, with a postulated regulatory role in STAT3 activation and G2/M transition. Interleukin-6, a major activator of STAT3, is present in elevated concentrations in uveal melanomas, suggesting contribution of dysregulated STAT3 activation to their pathogenesis. Here, we studied the impact of chelidonine on STAT3 signaling in human uveal melanoma cells. Chelidonine, an alkaloid isolated from Chelidonium majus, disrupts microtubules, causes mitotic arrest and provokes cell death in numerous tumor cells. According to our flow cytometry and confocal microscopy data, chelidonine abrogated IL-6-induced activation and nuclear translocation, but amplified constitutive serine phosphorylation of STAT3. Both effects were restricted to a fraction of cells only, in an all-or-none fashion. A partial overlap could be observed between the affected subpopulations; however, no direct connection could be proven. This study is the first proof on a cell-by-cell basis for the opposing effects of a microtubule-targeting agent on the two types of STAT3 phosphorylation.

Overexpression of pEGF improved the gut protective function of Clostridium butyricum partly through STAT3 signal pathway.

Clostridium butyricum (C. butyricum) is a probiotic that could promote animal growth and protect gut health. So far, current studies mainly keep up with the basic biological functions of C. butyricum, missing the effective strategy to further improve its protective efficiency. A recent report about C. butyricum alleviating intestinal injury through epidermal growth factor receptor (EGFR) inspired us to bridge this gap by porcine epidermal growth factor (EGF) overexpression. Lacking a secretory overexpression system, we constructed the recombinant strains overexpressing pEGF in C. butyricum for the first time and obtained 4 recombinant strains for highly efficient secretion of pEGF (BC/pPD1, BC/pSPP, BC/pGHF, and BC/pDBD). Compared to the wild-type strain, we confirmed that the expression level ranges of the intestinal development-related genes (Claudin-1, GLUT-2, SUC, GLP2R, and EGFR) and anti-inflammation-related gene (IL-10) in IPECs were upregulated under recombinant strain stimulation, and the growth of Staphylococcus aureus and Salmonella typhimurium was significantly inhibited as well. Furthermore, a particular inhibitor (stattic) was used to block STAT3 tyrosine phosphorylation, resulting in the downregulation on antibacterial effect of recombinant strains. This study demonstrated that the secretory overexpression of pEGF in C. butyricum could upregulate the expression level of EGFR, consequently improving the intestinal protective functions of C. butyricum partly following STAT3 signal activation in IPECs and making it a positive loop. These findings on the overexpression strains pointed out a new direction for further development and utilization of C. butyricum. KEY POINTS: • By 12 signal peptide screening in silico, 4 pEGF overexpression strains of C. butyricum/pMTL82151-pEGF for highly efficient secretion of pEGF were generated for the first time. • The secretory overexpression of pEGF promoted the intestinal development, antimicrobial action, and anti-inflammatory function of C. butyricum. • The overexpressed pEGF upregulated the expression level of EGFR and further magnified the gut protective function of recombinant strains which in turn partly depended on STAT3 signal pathway in IPECs.

Natural Trienoic Acids as Anticancer Agents: First Stereoselective Synthesis, Cell Cycle Analysis, Induction of Apoptosis, Cell Signaling and Mitochondrial Targeting Studies.

Simple SummaryCurrently, the whole world is acutely concerned with the selection of effective treatment regimens for oncological diseases. This problem is becoming more and more catastrophic every year due to the phenomenon of multidrug resistance, the consequence of which is the loss of the effectiveness of drugs against tumor cells. One of the solutions to the problem described above is the synthesis of new low molecular weight compounds that can effectively affect molecular cellular targets, for example, enzymes of the cell cycle, and, as a consequence, interrupt DNA synthesis, contributing to tumor death. Within the framework of this article, we carried out the Z-stereoselective synthesis of natural unsaturated acids containing a 1Z,5Z,9Z-triene moiety, for which it was shown that they are effective inhibitors of human topoisomerase I, and also affect mitochondria. At the same time, using multiplex analysis, the activation of signaling pathways was studied and a probable mechanism of the antitumor action of the synthesized trienoic acids was proposed.The first Z-stereoselective method was developed for the synthesis of unsaturated acids containing a 1Z,5Z,9Z-triene moiety in 61–64% yields using the new Ti-catalyzed cross-coupling of oxygen-containing and aliphatic 1,2-dienes as the key synthetic step. It was shown for the first time that trienoic acids with non-methylene-interrupted Z-double bonds show moderate cytotoxic activities against tumor cell lines (Jurkat, K562, U937, HL60, HeLa), human embryonic kidney cells (Hek293), normal fibroblasts and human topoisomerase I (hTop1) inhibitory activity in vitro. The synthesized acids efficiently initiate apoptosis of Jurkat tumor cells, with the cell death mechanism being activated by the mitochondrial pathway. A probable mechanism of topoisomerase I inhibition was also hypothesized on the basis of in silico studies resorting to docking. The activation and inhibition of the most versatile intracellular signaling pathways (CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3 and STAT5 tyrosine kinases) responsible for cell proliferation and for initiation of apoptosis were studied by multiplex assay technology (Luminex xMAP).

Brahma-Related Gene-1 (BRG1) promotes the malignant phenotype of glioblastoma cells.

Glioblastoma multiforme (GBM) is an aggressive malignant brain tumour that is resistant to existing therapeutics. Identifying signalling pathways deregulated in GBM that can be targeted therapeutically is critical to improve the present dismal prognosis for GBM patients. In this report, we have identified that the BRG1 (Brahma‐Related Gene‐1) catalytic subunit of the SWI/SNF chromatin remodelling complex promotes the malignant phenotype of GBM cells. We found that BRG1 is ubiquitously expressed in tumour tissue from GBM patients, and high BRG1 expression levels are localized to specific brain tumour regions. Knockout (KO) of BRG1 by CRISPR‐Cas9 gene editing had minimal effects on GBM cell proliferation, but significantly inhibited GBM cell migration and invasion. BRG1‐KO also sensitized GBM cells to the anti‐proliferative effects of the anti‐cancer agent temozolomide (TMZ), which is used to treat GBM patients in the clinic, and selectively altered STAT3 tyrosine phosphorylation and gene expression. These results demonstrate that BRG‐1 promotes invasion and migration, and decreases chemotherapy sensitivity, indicating that it functions in an oncogenic manner in GBM cells. Taken together, our findings suggest that targeting BRG1 in GBM may have therapeutic benefit in the treatment of this deadly form of brain cancer.

Stat3 oxidation-dependent regulation of gene expression impacts on developmental processes and involves cooperation with Hif-1α.

Reactive oxygen species are bona fide intracellular second messengers that influence cell metabolism and aging by mechanisms that are incompletely resolved. Mitochondria generate superoxide that is dis-mutated to hydrogen peroxide, which in turn oxidises cysteine-based enzymes such as phosphatases, peroxiredoxins and redox-sensitive transcription factors to modulate their activity. Signal Transducer and Activator of Transcription 3 (Stat3) has been shown to participate in an oxidative relay with peroxiredoxin II but the impact of Stat3 oxidation on target gene expression and its biological consequences remain to be established. Thus, we created murine embryonic fibroblasts (MEFs) that express either WT-Stat3 or a redox-insensitive mutant of Stat3 (Stat3-C3S). The Stat3-C3S cells differed from WT-Stat3 cells in morphology, proliferation and resistance to oxidative stress; in response to cytokine stimulation, they displayed elevated Stat3 tyrosine phosphorylation and Socs3 expression, implying that Stat3-C3S is insensitive to oxidative inhibition. Comparative analysis of global gene expression in WT-Stat3 and Stat3-C3S cells revealed differential expression (DE) of genes both under basal conditions and during oxidative stress. Using differential gene regulation pattern analysis, we identified 199 genes clustered into 10 distinct patterns that were selectively responsive to Stat3 oxidation. GO term analysis identified down-regulated genes to be enriched for tissue/organ development and morphogenesis and up-regulated genes to be enriched for cell-cell adhesion, immune responses and transport related processes. Although most DE gene promoters contain consensus Stat3 inducible elements (SIEs), our chromatin immunoprecipitation (ChIP) and ChIP-seq analyses did not detect Stat3 binding at these sites in control or oxidant-stimulated cells, suggesting that oxidised Stat3 regulates these genes indirectly. Our further computational analysis revealed enrichment of hypoxia response elements (HREs) within DE gene promoters, implying a role for Hif-1. Experimental validation revealed that efficient stabilisation of Hif-1α in response to oxidative stress or hypoxia required an oxidation-competent Stat3 and that depletion of Hif-1α suppressed the inducible expression of Kcnb1, a representative DE gene. Our data suggest that Stat3 and Hif-1α cooperate to regulate genes involved in immune functions and developmental processes in response to oxidative stress.

Loss of interleukin-17 receptor D promotes chronic inflammation-associated tumorigenesis.

Interleukin-17 receptor D (IL-17RD), also known as similar expression to Fgf genes (SEF), is proposed to act as a signaling hub that negatively regulates mitogenic signaling pathways, like the ERK1/2 MAP kinase pathway, and innate immune signaling. The expression of IL-17RD is downregulated in certain solid tumors, which has led to the hypothesis that it may exert tumor suppressor functions. However, the role of IL-17RD in tumor biology remains to be studied in vivo. Here, we show that genetic disruption of Il17rd leads to the increased formation of spontaneous tumors in multiple tissues of aging mice. Loss of IL-17RD also promotes tumor development in a model of colitis-associated colorectal cancer, associated with an exacerbated inflammatory response. Colon tumors from IL-17RD-deficient mice are characterized by a strong enrichment in inflammation-related gene signatures, elevated expression of pro-inflammatory tumorigenic cytokines, such as IL-17A and IL-6, and increased STAT3 tyrosine phosphorylation. We further show that RNAi depletion of IL-17RD enhances Toll-like receptor and IL-17A signaling in colon adenocarcinoma cells. No change in the proliferation of normal or tumor intestinal epithelial cells was observed upon genetic inactivation of IL-17RD. Our findings establish IL-17RD as a tumor suppressor in mice and suggest that the protein exerts its function mainly by limiting the extent and duration of inflammation.

Abstract B14: Atovaquone targets STAT3 in ovarian cancer spheroids

Abstract Ovarian cancer remains a deadly disease for countless women. Recent evidence demonstrates that ovarian cancer cell clusters, spheroids, are important in promoting ovarian recurrence and metastasis. Often these spheroids are resistant to therapy. Therefore, we were interested in identifying drugs that could target ovarian cancer spheroids. To do this, we wanted to identify pathways that were activated in cells growing in three-dimensional (3D) clusters. Analysis of gene expression from 2D and 3D growing cells identified STAT3 as a pathway enriched in 3D growing cells. STAT3 is a transcription factor that is activated by tyrosine phosphorylation. In ovarian cancer cells, activation usually occurs from interleukin 6 (IL-6) binding to the IL-6 receptor and recruiting the signal transducer molecule (GP130, IL6ST). This activates Jak kinases that then tyrosine phosphorylates STAT3. Once activated, STAT3 dimerizes, translocates into the nucleus, and regulates the expression of target genes. Using shRNA targeting STAT3 and the upstream signaling molecule, GP130, we found that reduction of both STAT3 and its upstream signaling molecule GP130 reduced the growth of spheroids. This suggested that targeting STAT3 or an upstream signaling molecule might be a viable treatment option. Atovaquone, used normally for the treatment of malaria or the prevention of pneumocystis pneumonia, inhibits STAT3 activation by inhibiting the expression of GP130 on the cell surface. Therefore, we hypothesized that atovaquone would have efficacy in ovarian cancer spheroids. Treatment of ovarian spheroids with atovaquone reduced their growth and viability. Importantly, atovaquone inhibited STAT3 tyrosine phosphorylation and target gene expression, demonstrating that atovaquone is inhibiting STAT3 in ovarian spheroids. Preliminary data suggest that overexpression of STAT3 partially rescues the effects of atovaquone on spheroid growth. As spheroids are also involved in ovarian cancer metastasis, we are currently assessing the effects of atovaquone on spheroid-mediated ovarian cancer metastasis. Taken together, our data suggest that atovaquone may be a beneficial treatment for ovarian cancer patients. Citation Format: Kayli E. Neil, Sarah J. Lacroix, David F. Walker, Suhu Liu, David A. Frank, Sarah R. Walker. Atovaquone targets STAT3 in ovarian cancer spheroids [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B14.

A77 1726, the active metabolite of the anti-rheumatoid arthritis drug leflunomide, inhibits influenza A virus replication in vitro and in vivo by inhibiting the activity of Janus kinases.

The newly reassorted IAV subtypes from zoonotic reservoirs respond poorly to current vaccines and antiviral therapy. There is an unmet need in developing novel antiviral drugs for better control of IAV infection. The cellular factors that are crucial for virus replication have been sought as novel molecular targets for antiviral therapy. Recent studies have shown that Janus kinases (JAK), JAK1, and JAK2, play an important role in IAV replication. Leflunomide is an anti-inflammatory drug primarily used for treating rheumatoid arthritis (RA). Prior studies suggest that A77 1726, the active metabolite of leflunomide, inhibits the activity of JAK1 and JAK3. Our current study aims to determine if A77 1726 can function as a JAK inhibitor to control IAV infection. Here, we report that A77 1726 inhibited the replication of three IAV subtypes（H5N1, H1N1, H9N2）in three cell types (chicken embryonic fibroblasts, A549, and MDCK). A77 1726 inhibited JAK1, JAK2, and STAT3 tyrosine phosphorylation. Similar observations were made with Ruxolitinib (Rux), a JAK-specific inhibitor. JAK2 overexpression enhanced H5N1 virus replication and compromised the antiviral activity of A77 1726. Leflunomide inhibited virus replication in the lungs of IAV-infected mice, alleviated their body weight loss, and prolonged their survival. Our study demonstrates for the first time the ability of A77 1726 to inhibit JAK2 activity and suggests that inhibition of JAK activity contributes to its antiviral activity.

IL-1β Enhances the Antiviral Effect of IFN-α on HCV Replication by Negatively Modulating ERK2 Activation.

Chronic hepatitis C infection is a leading cause of liver cirrhosis, which is linked to chronic hepatic inflammation. While there are multiple studies detailing the proinflammatory role of interleukin-1β (IL-1β) in HCV-induced inflammasome signaling, the antiviral capacity of this cytokine has not been adequately investigated in the context of HCV infection or other members of Flaviridae. Our data indicated that IL-1β alone does not inhibit HCV replication, yet when in combination with IFN-α, it can boost the anti-HCV activity of IFN-α, which is mediated by augmented STAT1 tyrosine 701 phosphorylation. Through signaling inhibitor screening, we found that ERK2 kinase is directly linked to the enhanced activation of the STAT1 complex. Our study found that IL-1β negatively affects ERK2 phosphorylation, which suggests that IL-1β-mediated STAT1 tyrosine 701 phosphorylation employed kinase machinery of ERK2 other than JNK or P38 kinase. Our results identify IL-1β as a proinflammatory cytokine possessing wide spectrum synergistic antiviral capability via enhancing IFN-α-induced interferon-stimulated genes (ISGs) expression. A more nuanced understanding of the antiviral mechanisms of this important cytokine could facilitate the development of new therapeutic options.

Pre-metastatic niche triggers SDF-1/CXCR4 axis and promotes organ colonisation by hepatocellular circulating tumour cells via downregulation of Prrx1

BackgroundCirculating tumour cells (CTCs), especially mesenchymal CTCs, are important determinants of metastasis, which leads to most recurrence and mortality in hepatocellular carcinoma (HCC). However, little is known about the underlying mechanisms of CTC colonisation in pre-metastatic niches.MethodsDetection and classification of CTCs in patients were performed using the CanPatrol™ system. A lentiviral vector expressing Prrx1-targeting shRNA was constructed to generate a stable HCC cell line with low expression of Prrx1. The effect of Prrx1 knockdown on stemness, migration, and drug resistance of the cell line was assessed, including involvement of SDF-1/CXCR4 signalling. Promising clinical applications of an inhibitor of STAT3 tyrosine phosphorylation, C188–9, and specific blockade with CXCR4 antibody were explored.ResultsThe number of mesenchymal CTCs in blood was closely associated with tumour recurrence or metastasis. Pre-metastatic niche-derived SDF-1 could downregulate Prrx1, which induced the stemness, drug resistance, and increased expression of CXCR4 in HCC cells through the STAT3 pathway in vitro. In vivo, mice bearing tumours of Prrx1 low-expressing cells had significantly shorter survival. In xenograft tumours and clinical samples, loss of Prrx1 was negatively correlated with increased expression of CXCR4 in lung metastatic sites compared with that in the primary foci.ConclusionsThese findings demonstrate that decreased expression of Prrx1 stimulates SDF-1/CXCR4 signalling and contributes to organ colonisation with blood CTCs in HCC. STAT3 inhibition and specific blockade of CXCR4 have clinical potential as therapeutics for eliminating organ metastasis in advanced HCC.