STAT3 Transcriptional Activity Research Articles

The transcriptional regulator Sin3A balances IL‐17A and Foxp3 expression in primary CD4 T cells

The Sin3 transcriptional regulator homolog A (Sin3A) is the core member of a multiprotein chromatin‐modifying complex. Its inactivation at the CD4/CD8 double‐negative stage halts further thymocyte development. Among various functions, Sin3A regulates STAT3 transcriptional activity, central to the differentiation of Th17 cells active in inflammatory disorders and opportunistic infections. To further investigate the consequences of conditional Sin3A inactivation in more mature precursors and post‐thymic T cell, we have generated CD4‐Cre and CD4‐CreERT2 Sin3AF/F mice. Sin3A inactivation in vivo hinders both thymocyte development and peripheral T‐cell survival. In vitro, in Th17 skewing conditions, Sin3A‐deficient cells proliferate and acquire memory markers and yet fail to properly upregulate Il17a, Il23r, and Il22. Instead, IL‐2+ and FOXP3+ are mostly enriched for, and their inhibition partially rescues IL‐17A+ T cells. Notably, Sin3A deletion also causes an enrichment of genes implicated in the mTORC1 signaling pathway, overt STAT3 activation, and aberrant cytoplasmic RORγt accumulation. Thus, together our data unveil a previously unappreciated role for Sin3A in shaping critical signaling events central to the acquisition of immunoregulatory T‐cell phenotypes.

Acute respiratory distress syndrome enhances tumor metastasis into lungs: Role of BRD4 in the tumor microenvironment.

Acute respiratory distress syndrome (ARDS) is associated with severe lung inflammation, edema, hypoxia, and high vascular permeability. The COVID-19-associated pandemic ARDS caused by SARS-CoV-2 has created dire global conditions and has been highly contagious. Chronic inflammatory disease enhances cancer cell proliferation, progression, and invasion. We investigated how acute lung inflammation activates the tumor microenvironment and enhances lung metastasis in LPS induced in vitro and in vivo models. Respiratory illness is mainly caused by cytokine storm, which further influences oxidative and nitrosative stress. The LPS-induced inflammatory cytokines made the conditions suitable for the tumor microenvironment in the lungs. In the present study, we observed that LPS induced the cytokine storm and promoted lung inflammation via BRD4, which further caused the nuclear translocation of p65 NF-κB and STAT3. The transcriptional activation additionally triggers the tumor microenvironment and lung metastasis. Thus, BRD4-regulated p65 and STAT3 transcriptional activity in ARDS enhances lung tumor metastasis. Moreover, LPS-induced ARDS might promote the tumor microenvironment and increase cancer metastasis into the lungs. Collectively, BRD4 plays a vital role in inflammation-mediated tumor metastasis and is found to be a diagnostic and molecular target in inflammation-associated cancers.

Cycloguanil and Analogues Potently Target DHFR in Cancer Cells to Elicit Anti-Cancer Activity.

Dihydrofolate reductase (DHFR) is an established anti-cancer drug target whose inhibition disrupts folate metabolism and STAT3-dependent gene expression. Cycloguanil was proposed as a DHFR inhibitor in the 1950s and is the active metabolite of clinically approved plasmodium DHFR inhibitor Proguanil. The Cycloguanil scaffold was explored to generate potential cancer therapies in the 1970s. Herein, current computational and chemical biology techniques were employed to re-investigate the anti-cancer activity of Cycloguanil and related compounds. In silico modeling was employed to identify promising Cycloguanil analogues from NCI databases, which were cross-referenced with NCI-60 Human Tumor Cell Line Screening data. Using target engagement assays, it was found that these compounds engage DHFR in cells at sub-nanomolar concentrations; however, growth impairments were not observed until higher concentrations. Folinic acid treatment rescues the viability impairments induced by some, but not all, Cycloguanil analogues, suggesting these compounds may have additional targets. Cycloguanil and its most promising analogue, NSC127159, induced similar metabolite profiles compared to established DHFR inhibitors Methotrexate and Pyrimethamine while also blocking downstream signaling, including STAT3 transcriptional activity. These data confirm that Cycloguanil and its analogues are potent inhibitors of human DHFR, and their anti-cancer activity may be worth further investigation.

STAT3 mutation-associated airway epithelial defects in Job syndrome

Job syndrome is a disease of autosomal dominant hyper-IgE syndrome (AD-HIES). Patients harboring STAT3 mutation are particularly prone to airway remodeling and airway infections. Airway epithelial cells play a central role as the first line of defense against pathogenic infection and express high levels of STAT3. This study thus interrogates how AD-HIES STAT3 mutations impact the physiological functions of airway epithelial cells. This study created human airway basal cells expressing 4 common AD-HIES STAT3 mutants (R382W, V463del, V637M, and Y657S). In addition, primary airway epithelial cells were isolated from a patient with Job syndrome who was harboring a STAT3-S560del mutation and from mice harboring a STAT3-V463del mutation. Cell proliferation, differentiation, barrier function, bacterial elimination, and innate immune responses to pathogenic infection were quantitatively analyzed. STAT3 mutations reduce STAT3 protein phosphorylation, nuclear translocation, transcription activity, and protein stability in airway basal cells. As a consequence, STAT3-mutated airway basal cells give rise to airway epithelial cells with abnormal cellular composition and loss of coordinated mucociliary clearance. Notably, AD-HIES STAT3 airway epithelial cells are defective in bacterial killing and fail to initiate vigorous proinflammatory responses and neutrophil transepithelial migration in response to an experimental model of Pseudomonas aeruginosa infection. AD-HIES STAT3 mutations confer numerous abnormalities to airway epithelial cells in cell differentiation and host innate immunity, emphasizing their involvement in the pathogenesis of lung complications in Job syndrome. Therefore, therapies must address the epithelial defects as well as the previously noted immune cell defects to alleviate chronic infections in patients with Job syndrome.

Homocysteine causes neuronal leptin resistance and endoplasmic reticulum stress.

Abnormally high serum homocysteine levels have been associated with several disorders, including obesity, cardiovascular diseases or neurological diseases. Leptin is an anti-obesity protein and its action is mainly mediated by the activation of its Ob-R receptor in neuronal cells. The inability of leptin to induce activation of its specific signaling pathways, especially under endoplasmic reticulum stress, leads to the leptin resistance observed in obesity. The present study examined the effect of homocysteine on leptin signaling in SH-SY5Y neuroblastoma cells expressing the leptin receptor Ob-Rb. Phosphorylation of the signal transducer and activator of transcription (STAT3) and leptin-induced STAT3 transcriptional activity were significantly inhibited by homocysteine treatment. These effects may be specific to homocysteine and to the leptin pathway, as other homocysteine-related compounds, namely methionine and cysteine, have weak effect on leptin-induced inhibition of STAT3 phosphorylation, and homocysteine has no impact on IL-6-induced activation of STAT3. The direct effect of homocysteine on leptin-induced Ob-R activation, analyzed by Ob-R BRET biosensor to monitor Ob-R oligomerization and conformational change, suggested that homocysteine treatment does not affect early events of leptin-induced Ob-R activation. Instead, we found that, unlike methionine or cysteine, homocysteine increases the expression of the endoplasmic reticulum (ER) stress response gene, a homocysteine-sensitive ER resident protein. These results suggest that homocysteine may induce neuronal resistance to leptin by suppressing STAT3 phosphorylation downstream of the leptin receptor via ER stress.

PITX1 suppresses osteosarcoma metastasis through exosomal LINC00662-mediated M2 macrophage polarization

Paired-like homeodomain transcription factor 1 (PITX1) is frequently downregulated in cancers, including osteosarcoma (OS). However, its role in OS remains unknown. Therefore, we aimed to explore the functions and potential mechanisms of PITX1 in OS malignant progression. Elevated PITX1 suppressed OS cell proliferation and migration, based on transwell, proliferation, and colony formation assays. Pathway enrichment analysis of differentially-expressed genes between PITX1-overexpressing and control OS cells indicated that PITX1 expression was associated with the FAK/Src and PI3k/Akt signaling pathways. Mechanistically, ubiquitination assays and rescue experiments showed that PITX1 interacted with transcription factor STAT3, leading to decreased STAT3 transcriptional activity, which repressed the expression of LINC00662. Specific knockdown of LINC00662 reduced the tumor growth and invasion of OS cells induced by downregulated PITX1. Moreover, exosomal LINC00662, derived from PITX1 knockdown OS cell lines activated M2 macrophages in cell co-culture assays. M2 macrophage secreted several cytokines, among which CCL22 was found to cause OS cell EMT. Collectively, our data indicate that PITX1 suppresses OS cell proliferation and metastasis by downregulating LINC00662. Moreover, LINC00662 can be packaged into OS cell-derived exosomes to mediate M2 macrophage polarization to promote OS metastasis via CCL22.

Loss of LXN promotes macrophage M2 polarization and PD-L2 expression contributing cancer immune-escape in mice

Latexin (LXN) plays an important role in tumorigenesis and inflammatory response and as a tumor suppressor in many tumors. However, whether LXN regulates tumorigenesis through immune regulation remains uncertain. Here, we demonstrate that LXN deficiency increases hematopoietic stem cells, as well as affects the proportion of immune cells in the peripheral system. Animal studies show that mice loss of LXN promotes tumor growth in subcutaneous tumor model and AOM/DSS-induced colorectal cancer model. We found that loss of LXN promotes macrophage M2 polarization and PD-L2 expression in macrophage, thus, inhibits the function of T cells. Adoptive transfer of wild-type macrophage rescues the function of T cells in LXN-deficient mice. LXN deficiency in hematopoietic lineage exacerbates colorectal carcinogenesis, and targeted inhibition of PD-L2 ameliorates cancer growth in LXN-deficient mice. Mechanistically, we demonstrate that LXN inhibits STAT3 transcriptional activity by targeting inhibition of JAK1 in macrophages. LXN deficiency enhances PD-L2 expression rather than PD-L1 in macrophages, which lead to inhibition of T cells in tumor microenvironment. Collectively, we define a critical role of LXN/JAK1/STAT3 signal in macrophage and highlights the potential role of LXN in tumor immune-escape by regulating macrophage polarization, as well as the expression of immune checkpoint PD-L2.

Targeting the transcriptional activity of STAT3 by a novel fusion protein

BackgroundThe continuous activation of transcription factors drives many diseases, including tumors, autoimmune disease, neurodegenerative disease, and male infertility. Thus, Blocking the transcriptional activity of these proteins may inhibit disease progression. In this study, we developed a new method to specifically inhibit the activity of the transcription factor STAT3.MethodsFusing the transcriptional inhibitory domain KRAB with STAT3 successfully blocked the transcription activity of STAT3 in cancer cells without affecting its function in the mitochondria and lysosomes.Resultsthe expression of KRAB-STAT3 fusion protein inhibited the growth of tumor cells.ConclusionsThe KRAB-STAT3 fusion protein provides a novel approach for drug development for the treatment of cancer or autoimmune diseases.

Abstract 2364: ABI1 regulates transcriptional activity of STAT3

Abstract Background: Prostate cancer (PCa) is characterized by the complexity of oncogenic signaling and heterogeneity of transcriptional landscapes. Adaptor protein ABI1 is a tumor suppressor in PCa as evidenced by its loss or downregulation in high grade and metastatic tumors. STAT3 activation is a hallmark of high-risk prostate tumors. ABI1 loss is associated with STAT3 activation leading to transcriptional reprogramming and epithelial-mesenchymal-transition (EMT) of prostate cancer cells (Nath, Li et al. 2019 Cell Commun Signal). EMT changes involve several homeobox transcription factors. The fact that ABI1 contains a homeobox homology region (HHR) suggested the possibility that it plays a role in EMT through directly regulating transcriptional activity by DNA binding. Methods: To examine this hypothesis we set out to analyze ABI1-DNA binding. We purified ABI1 HHR and demonstrated its in vitro interactions with different homeobox DNA-binding consensus of double-stranded DNA sequences. We created DU145 ABI1 CRISPR KO cell line and rescue ABI1 expression using retroviral transfection with either ABI1 wild type or Abi1 HHR mutants. DU145 cell lines STAT3 cellular localization was studied by Immunofluorescence staining and the STAT3 transcription activities was studied by Firefly Luciferase Assays with STAT3 specific luciferase reporter plasmid. Results: We found that the presence of alternatively spliced Exon 4-encoded sequence, located in the C-terminus of HHR, regulates binding of ABI1 to DNA. Structural NMR studies confirmed ABI1 binding to DNA. Subsequent functional studies using DU145 CRISPR KO cell lines expressing wild type or HHR mutants of ABI1 demonstrated that HHR regulates the nuclear localization and transcriptional activity of STAT3. Conclusion: We propose that ABI1 is a critical regulator of STAT3 activity during prostate cancer progression by the ABI1 HHR mediated DNA binding mechanism. Funding resources: NCI R01 CA161018; R21 CA260381 Conflicts of Interest Disclosure Statement: The authors declare no conflicts of interest. Citation Format: Xiang Li, Baylee Porter, Allysa Kemraj, Alaji Bah, Marcin Kortylewski, Leszek Kotula. ABI1 regulates transcriptional activity of STAT3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2364.

Abstract 1404: Novel STAT3 inhibitors targeting the STAT3 dimerization

Abstract Background The STAT3 pathway may drive prostate cancer (PCa) progression to metastatic castration-resistant prostate cancer (mCRPC). STAT3 may serve as a good target for the treatment of prostate cancer. The function of STAT3 relies significantly on its SH2 domain, which promotes STAT3 homo- or hetero-dimerization, protein-protein interaction and nuclear translocation of the STAT3 dimers needed for transcription. Due to this important role, the STAT3 SH2 domain becomes an attractive therapeutic target. In a drug discovery and development program we have found two small molecular compounds named 323-1 and 323-2 that were identified as novel STAT3 SH2 domain inhibitors in a series of experiments. Material and methods In silico computational modeling, Drug Affinity Responsive Target Stability (DARTS) and Fluorescence Polarization (FP) assays were performed to examine whether compounds 323-1 and 323-2 could bind to the STAT3 protein. The antitumor activity of compounds 323-1 and 323-2 was firstly determined by clonogenic assays in different prostate tumor cells, then validated in the humanised NOD/SCID mouse CRPC model. Effects of compound 323-1 and 323-2 on STAT3 transcriptional activity were determined by the luciferase assay and protein levels by western blotting. Results In silico computational modeling, DARTS and FP assays altogether determined 323-1 and 323-2 as direct STAT3 inhibitors targeting the STAT3 SH2 domain and inhibiting both phosphorylated and non-phosphorylated STAT3 dimerization. Computational docking predicted that both 323 compounds bind to three subpockets of the STAT3 SH2 domain with full inhibition of STAT3. FP assay further confirmed that 323s target the STAT3 SH2 domain by competitively abrogating the interaction between STAT3 and the SH2-binding peptide GpYLPQTV. Compared with S3I-201, the 323 compounds exhibited stronger inhibition of STAT3 and reduced the level of IL-6-stimulated phosphorylation of STAT3 (Tyr705) in LNCaP cells over the phosphorylation of STAT1 (Tyr701) induced by IFN-ɣ in PC3 cells or the phosphorylation of STAT1 (Ser727) in DU145 cells. Both compounds down-regulated STAT3 target genes MCL1 and cyclin D1. The in-vivo data supported that 323s significantly inhibited LNCaP cell generated CRPC tumor growth. Thus, the two compounds are promising leading compounds for the treatment of cancers with hyper-activated STAT3. Conclusions Here we report that these compounds modulate the IL-6/STAT3 pathway by 1) inhibition of STAT3 phosphorylation on Tyr705 and 2) disruption of STAT3 dimerization by directly targeting its SH2 domain; 3) inhibition of STAT3 transcriptional activity. Thus, the compounds 323-1 and 323-2 are promising new leading compounds for therapeutic STAT3 inhibition. Citation Format: Yaping Hua, Waqas Azeem, Anne Margrete Øyan, Karl-Henning Kalland. Novel STAT3 inhibitors targeting the STAT3 dimerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1404.

Abstract 1689: Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts

Abstract Classical markers of tumor-initiating cells (TIC), aldehyde dehydrogenase 1 (ALDH1) activity and the cell-surface markers, CD44pos/CD24low/neg, show insufficient cell type specificity to identify TIC definitively, nor do they allow robust enrichment by FACS. As a consequence, analyses of TIC function and gene expression to date are not definitive. Identification and characterization of the TIC subpopulation is critically important in breast cancer since these cells are thought to mediate treatment resistance, as well as local and distant recurrence. This is particularly critical for “triple negative” breast cancer (TNBC), since currently there are no targeted therapies available for this clinical subtype. Lentiviral signaling reporter vectors allow purification of enriched TIC subpopulations thereby facilitating single cell analyses to identify distinct sets of cells with TIC features. We used a collection of fluorescent lentiviral reporters that enable visualization and flow cytometric enrichment of cells undergoing STAT3-, GLI-, WNT-, or OCT4/SOX2-mediated transcriptional activity. Our collection of patient derived xenograft (PDX) models of TNBC were infected with the lentiviral reporters, re-transplanted, and screened for reporter activity upon regrowth. TIC function of reporter-expressing cells relative to negative cells was assayed by mammosphere formation and limiting dilution transplantation. Reporter expressing cells with enriched TIC function were then analyzed by single cell RNAseq for expression of classical markers to identify the subset of cells most likely to represent the TIC subpopulation. Our results indicate that STAT3 and GLI transcriptional activity can serve as uniquely PDX-dependent indicators for TIC function for a subpopulation of cancer cells. Also, multiple potential TIC signaling pathways, including STAT3, GLI, WNT, and OCT4/SOX2, can be active in a single PDX model, but do not necessarily associate with TIC function, indicating TIC diversity in TNBC. These data in conjunction with treatment response and single-cell RNA-seq sheds light on the molecular diversity of TIC populations in TNBC, identifies candidate targets for therapeutic intervention, and will indicate whether TIC diversity is associated with differential response to different chemotherapeutic agents. Citation Format: John Landua, Ping Gong, Michael T. Lewis. Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1689.

HIPK2 directs cell type–specific regulation of STAT3 transcriptional activity in Th17 cell differentiation

SignificanceSTAT3 (signal transducer and activator of transcription 3) is a master transcription factor that organizes cellular responses to cytokines and growth factors and is implicated in inflammatory disorders. STAT3 is a well-recognized therapeutic target for human cancer and inflammatory disorders, but how its function is regulated in a cell type-specific manner has been a major outstanding question. We discovered that Stat3 imposes self-directed regulation through controlling transcription of its own regulator homeodomain-interacting protein kinase 2 (Hipk2) in a T helper 17 (Th17) cell-specific manner. Our validation of the functional importance of the Stat3-Hipk2 axis in Th17 cell development in the pathogenesis of T cell-induced colitis in mice suggests an approach to therapeutically treat inflammatory bowel diseases that currently lack a safe and effective therapy.

Zebrafish Mutant Lines Reveal the Interplay between nr3c1 and nr3c2 in the GC-Dependent Regulation of Gene Transcription.

Glucocorticoids mainly exert their biological functions through their cognate receptor, encoded by the nr3c1 gene. Here, we analysed the glucocorticoids mechanism of action taking advantage of the availability of different zebrafish mutant lines for their receptor. The differences in gene expression patterns between the zebrafish gr knock-out and the grs357 mutant line, in which a point mutation prevents binding of the receptor to the hormone-responsive elements, reveal an intricate network of GC-dependent transcription. Particularly, we show that Stat3 transcriptional activity mainly relies on glucocorticoid receptor GR tethering activity: several Stat3 target genes are induced upon glucocorticoid GC exposure both in wild type and in grs357/s357 larvae, but not in gr knock-out zebrafish. To understand the interplay between GC, their receptor, and the mineralocorticoid receptor, which is evolutionarily and structurally related to the GR, we generated an mr knock-out line and observed that several GC-target genes also need a functional mineralocorticoid receptor MR to be correctly transcribed. All in all, zebrafish mutants and transgenic models allow in vivo analysis of GR transcriptional activities and interactions with other transcription factors such as MR and Stat3 in an in-depth and rapid way.

TRIM32 promotes radioresistance by disrupting TC45-STAT3 interaction in triple-negative breast cancer.

Radioresistance is common in the treatment of triple-negative breast cancer (TNBC), but the molecular mechanisms involved remain unclear. Herein, we reveal that tripartite motif-containing protein 32 (TRIM32) is upregulated in TNBC and is negatively associated with survival of TNBC patients. Radiotherapy resulted in enhanced expression of TRIM32, whereas TRIM32 depletion reduced TNBC radioresistance in vitro and in vivo. Mechanistically, radiotherapy promoted the association between TRIM32 and nuclear STAT3, which suppressed TC45-induced dephosphorylation of STAT3, resulting in increased STAT3 transcriptional activation and TNBC radioresistance. Finally, we demonstrated that TRIM32 and STAT3 phosphorylation are co-expressed in TNBC tissues. Moreover, high expression of TRIM32 and STAT3 phosphorylation is positively linked to poor prognosis of TNBC patients. Our study demonstrates that TRIM32 is a novel target for predicting radioresistance in TNBC patients.

Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFR\u03b2 and affect PDGF-BB-induced STAT3 signalling

Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor β (PDGFRβ) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRβ did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRβ lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.

STAT3 Signaling in Breast Cancer: Multicellular Actions and Therapeutic Potential.

Simple SummaryMany signaling pathways are overactive in breast cancer, and among them is the STAT3 signaling pathway. STAT3 is activated by secreted factors within the breast tumor, many of which are elevated and correlate to advanced disease and poor survival outcomes. This review examines how STAT3 signaling is activated in breast cancer by the proinflammatory, gp130 cytokines, interleukins 6 and 11. We evaluate how this signaling cascade functions in the various cells of the tumor microenvironment to drive disease progression and metastasis. We discuss how our understanding of these processes may lead to the development of novel therapeutics to tackle advanced disease.Interleukin (IL)-6 family cytokines, such as IL-6 and IL-11, are defined by the shared use of the gp130 receptor for the downstream activation of STAT3 signaling and the activation of genes which contribute to the “hallmarks of cancer”, including proliferation, survival, invasion and metastasis. Increased expression of these cytokines, or the ligand-specific receptors IL-6R and IL-11RA, in breast tumors positively correlate to disease progression and poorer patient outcome. In this review, we examine evidence from pre-clinical studies that correlate enhanced IL-6 and IL-11 mediated gp130/STAT3 signaling to the progression of breast cancer. Key processes by which the IL-6 family cytokines contribute to the heterogeneous nature of breast cancer, immune evasion and metastatic potential, are discussed. We examine the latest research into the therapeutic targeting of IL-6 family cytokines that inhibit STAT3 transcriptional activity as a potential breast cancer treatment, including current clinical trials. The importance of the IL-6 family of cytokines in cellular processes that promote the development and progression of breast cancer warrants further understanding of the molecular basis for its actions to help guide the development of future therapeutic targets.

Deploying Kinase Inhibitors to Study Pediatric Gliomas.

Pediatric midline gliomas are a uniformly fatal disease for which there is no cure. The location of these tumors makes surgical resection impossible, and so novel therapies are urgently needed to improve outcomes. The biology of these tumors is increasingly understood, with the histone H3K27M mutation playing a critical role in the pathogenesis of these tumors. Efforts to inhibit the growth of these tumors have also focused on inhibiting the Aurora kinase and Janus-associated kinase (JAK)/signal transducer and activator of transcription (STAT) pathway in order to disrupt tumor proliferation. A number of small molecule inhibitors of these kinases have shown promise in early studies. Screening and preclinical assessment of such inhibitors requires a functional assay to assess the degree of kinase inhibition. We detail here a luciferase-based reporter assay for STAT3 transcriptional activity that we have employed frequently in order to assess the efficacy of kinase inhibitors in pediatric gliomas. The assay we describe is specific to STAT3, but the overall methodology is generalizable to other downstream targets of the kinase of interest.

The Mechanism Underlying the Inverse Proportion between Serum IgE Concentration and Stat3 Transcriptional Activity Depends

The Mechanism Underlying the Inverse Proportion between Serum IgE Concentration and Stat3 Transcriptional Activity Depends

Ethyl Acetate Fraction in Hedyotis Diffusa Willd Inhibits T Cell Proliferation to Improve the Pathogenesis of Systemic Lupus Erythematosus

Abnormal proliferation of T cells plays an essential role in the pathogenesis of Systemic lupus erythematosus (SLE). The pharmaceutical effect of Hedyotis Diffusa Willd (HDW) on SLE has been investigated previously. Nevertheless, the biomedical mechanism is still left unclear. This study has been arranged to evaluate the therapeutic effect of the ethyl acetate fraction of HDW (EAHDW) on lupus mice and explore the potential therapeutic mechanism. EAHDW was prepared with 80% ethanol reflex extraction followed by successive extraction, and analyzed with HPLC and UPLC-Q/TOP-MS. The potential targets and STAT3 affinity regulators were predicted with network pharmacology. The pharmaceutic effect of EAHDW was studied with MRL/lpr mice. Cytokines and autoantibodies were quantified with ELISA assays. The pathological damage of glomerulus and STAT3 expression in the kidney was detected with histochemical and immunohistochemical techniques. The cell cycle properties in cell proliferation were identified with the flow cytometry. The western blot and dual-Luciferase reporter assay were applied to evaluate translational and transcriptional activity of STAT3, respectively. In this study, the extraction ratio of EAHDW was 2.7±1%, in which 19 ingredients were identified. Network pharmacological analysis showed that the target genes of EAHDW were highly focused on influencing the abnormal T cell proliferation in SLE. EAHDW showed the beneficial effects on pathological changes and STAT3 expression in the glomerulus of lupus mice, and the levels of cytokines and autoantibodies in serum. In cytological study, EAHDW treatment attenuated the transcription and phosphorylation of STAT3, which inhibited T cell proliferation by prolonged S-phase of the cell cycle. A total of 5 compounds in EAHDW exhibited high docking affinity to the DNA-binding site of STAT3. EAHDW could reduce the inflammatory response and inhibit the proliferation of T cells by interfering with the STAT3 signaling pathway, thereby playing a therapeutic effect on SLE.

An ELISA-based method for rapid genetic screens in Drosophila

Drosophila is a powerful model in which to perform genetic screens, but screening assays that are both rapid and can be used to examine a wide variety of cellular and molecular pathways are limited. Drosophila offer an extensive toolbox of GFP-based transcriptional reporters, GFP-tagged proteins, and driver lines, which can be used to express GFP in numerous subpopulations of cells. Thus, a tool that can rapidly and quantitatively evaluate GFP levels in Drosophila tissue would provide a broadly applicable screening platform. We developed a GFP-based enzyme-linked immunosorbent assay (ELISA) that can detect GFP in Drosophila lysates collected from whole animals and dissected tissues across all stages of Drosophila development. We demonstrate that this assay can detect membrane-localized GFP in a variety of neuronal and glial populations and validate that it can identify genes that change the morphology of these cells, as well as changes in STAT and JNK transcriptional activity. We found that this assay can detect endogenously GFP-tagged proteins, including Draper, Cryptochrome, and the synaptic marker Brp. This approach is able to detect changes in Brp-GFP signal during developmental synaptic remodeling, and known genetic regulators of glial synaptic engulfment could be identified using this ELISA method. Finally, we used the assay to perform a small-scale screen, which identified Syntaxins as potential regulators of astrocyte-mediated synapse elimination. Together, these studies establish an ELISA as a rapid, easy, and quantitative in vivo screening method that can be used to assay a wide breadth of fundamental biological questions.