Signal Transducer And Activator Of Transcription 3 Target Research Articles

Abstract 417: Targeting aurora kinase b overcomes acquired resistance to met-tyrosine kinase inhibitor in met-amplified lung cancer

Abstract Mesenchymal-epithelial transition factor receptor (MET) mediated signaling emerged as an important clinical target for the treatment of many cancer types including non-small cell lung cancer. MET amplification is prevalent in 2%~4% of primary lung cancer and 15%~50% of EGFR-mutated lung cancer resistant to EGFR inhibitors. Currently, MET-targeting drugs have been actively investigated in these MET-amplified lung cancers. However, the acquired resistance mechanism for MET-targeted therapy remains unknown. Our study aims to provide effective treatment for overcoming MET-TKI-acquired resistance in MET-amplified non-small cell lung cancer. To discover the resistance mechanism, we established the resistant cell line (PR-S2) by chronic exposure of MET-amplified lung cancer cells (H1993) to MET kinase inhibitor, PHA665752. Through high-throughput screening of a custom library of 276 compounds, we found that Aurora kinase B (AURKB) inhibitor, barasertib was more sensitive to the PR-S2 cells than H1993 cells. AURKB inhibition by barasertib induced apoptotic cell death with G2/M arrest and polyploid cell formation in the PR-S2 cells. The expression of p-AURKB was increased with no change in the expression of p-histone H3, a major product of AURKB in the PR-S2 cells. AURKB knockdown using multiple siRNA sequences increases the sensitivity to PHA665752 in the PR-S2 cells. In mRNA sequencing data analysis, we found the expression of signal transducer and activator of transcription 3 (STAT3) was elevated in the PR-S2 cells compared to the H1993 cells. We confirmed increased STAT3 expression in both protein and mRNA levels in the PR-S2 cells. p-STAT3 expression was decreased after barasertib treatment in the PR-S2 cells. However, in the immunoprecipitation assay, there was no direct interaction between AURKB and STAT3. To know the target of STAT3, we checked the mRNA expression of downstream genes of STAT3. The expression of BCL-2, one of the downstream targets of STAT3 and the central apoptosis regulator, was highly upregulated in the resistant cells. The siRNA-mediated knockdown of STAT3 expression decreased BCL2 expression in the PR-S2 cells, whereas not in the H1993 cells. Furthermore, we investigated apoptosis-related gene expressions and found that the BH3-only proapoptotic member of the BCL-2 protein family, BIM was significantly decreased in the PR-S2 cells. AURKB inhibition induced BIMEL accumulation and reduced p-BIM expression. Collectively, our study suggests that AURKB activation induces resistance to MET-TKI through anti-apoptosis mechanism with upregulation of STAT3/BCL2 axis in MET-amplified lung cancer cells. Citation Format: Yura Choi, Mihwa Hwang, Sunshin Kim, Jae J. Song, Youngjoo Lee. Targeting aurora kinase b overcomes acquired resistance to met-tyrosine kinase inhibitor in met-amplified lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 417.

Abstract 1587: Breast cancer treatment: Exploring bazedoxifene, a known SERM, SERD and indirect STAT3 inhibitor

Abstract In 2020, breast cancer was the most widespread cancer. This heterogenous disease can be divided into molecular subtypes according to three biomarkers: Estrogen Receptor (ER), Progesterone Receptor (PR) and Epidermal Growth Factor Receptor 2 (HER2). Currently, triple-negative breast cancers (TNBC), negative for all three biomarkers, have no personalized treatment. Also, about 40% of women with breast cancer receiving tamoxifen, a selective estrogen receptor modulator (SERM), have or will develop a resistance. Bazedoxifene (BZA), a SERM, a selective estrogen receptor modulator (SERD) and an indirect Signal transducer and activator of transcription 3 (STAT3) inhibitor, used for treatment of osteoporosis, has exhibited in vitro antitumoral effects. Therefore, BZA efficacy must be further evaluated. We hypothesize that BZA is a therapeutic option for hormone receptor (HR)-positive breast cancers and a novel anticancer agent for TNBC patients. To verify our hypothesis, we performed 2D viability assays on breast cancer cell lines treated with different BZA and hydroxytamoxifen (4OHT) concentrations to calculate the absolute half maximal inhibitory concentration (IC50). We then performed 3D viability assays, which is a first with BZA treatment. We further investigated gene expression of known STAT3 targets in breast cancer subtypes in UALCAN and on survival outcomes in PRECOG and ROCplot. The 72 hours BZA IC50 is respectively 8.038 ± 0.132 µM for TNBC basal-like 2 cell line (SUM149PT) and the 4OHT IC50 for this cell line is 6.095 ± 0.133 µM (p-value= 0.1). Despite IC50 seeming lower for 4OHT, the results are not significant when performing two sided Mann-Whitney. Similarly, for MDA-MB-231, another TNBC cell line, characterized as mesenchymal-like, BZA IC50 (7.988± 0.482 µM) seems higher but is not significantly different to the 4OHT IC50 (7.080± 0.167 µM) (p-value= 0.1). We also found that BZA IC50 and 4OHT IC50 for MCF7 (HR+) are not significantly different (p-value= 0.4). 3D viability assays performed on SUM149PT spheroids treated for 3 days with 2X and 5X BZA IC50 decreased cell viability whilst 2X 4OHT IC50 did not reduce cell viability. Lastly, we determined that BIRC5, a known inhibitor of apoptosis, is more expressed in TNBC patients from TCGA breast cancer cohort than other molecular subtypes, but patients diagnosed with luminal A breast cancer (HR+/HER2-) receiving chemotherapy and experiencing a recurrence have a higher median gene expression of BIRC5. In summary, we have shown that BZA seems less effective to reduce cell viability in 2D assays than 4OHT in TNBC cell line but may be more effective in 3D models. A decrease in phosphorylated STAT3 following BZA treatment could be beneficial to patients with luminal A breast cancer who are not responding to chemotherapy. Citation Format: Juliette Bherer, Alisson Clemenceau, Caroline Diorio, Francine Durocher. Breast cancer treatment: Exploring bazedoxifene, a known SERM, SERD and indirect STAT3 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1587.

STAT3 is Activated by CTGF-mediated Tumor-stroma Cross Talk to Promote HCC Progression.

Signal transducer and activator of transcription 3 (STAT3) is known as a pro-oncogenic transcription factor. Regarding liver carcinogenesis, however, it remains controversial whether activated STAT3 is pro- or anti-tumorigenic. This study aimed to clarify the significance and mechanism of STAT3 activation in hepatocellular carcinoma (HCC). Hepatocyte-specific Kras-mutant mice (Alb-Cre KrasLSL-G12D/+; KrasG12D mice) were used as a liver cancer model. Cell lines of hepatoma and stromal cells including stellate cells, macrophages, T cells, and endothelial cells were used for culture. Surgically resected 12 HCCs were used for human analysis. Tumors in KrasG12D mice showed up-regulation of phosphorylated STAT3 (p-STAT3), together with interleukin (IL)-6 family cytokines, STAT3 target genes, and connective tissue growth factor (CTGF). Hepatocyte-specific STAT3 knockout (Alb-Cre KrasLSL-G12D/+ STAT3fl/fl) downregulated p-STAT3 and CTGF and suppressed tumor progression. In coculture with stromal cells, proliferation, and expression of p-STAT3 and CTGF, were enhanced in hepatoma cells via gp130/STAT3 signaling. Meanwhile, hepatoma cells produced CTGF to stimulate integrin/nuclear factor kappa B signaling and up-regulate IL-6 family cytokines from stromal cells, which could in turn activate gp130/STAT3 signaling in hepatoma cells. In KrasG12D mice, hepatocyte-specific CTGF knockout (Alb-Cre KrasLSL-G12D/+ CTGFfl/fl) downregulated p-STAT3, CTGF, and IL-6 family cytokines, and suppressed tumor progression. In human HCC, single cell RNA sequence showed CTGF and IL-6 family cytokine expression in tumor cells and stromal cells, respectively. CTGF expression was positively correlated with that of IL-6 family cytokines and STAT3 target genes in The Cancer Genome Atlas. STAT3 is activated by CTGF-mediated tumor-stroma crosstalk to promote HCC progression. STAT3-CTGF positive feedback loop could be a therapeutic target.

Inhibition of deubiquitinase USP28 attenuates cyst growth in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, which is characterized by progressive growth of multiple renal cysts in bilateral kidneys. In the past decades, mechanistic studies have entailed many essential signalling pathways that were regulated through post-translational modifications (PTMs) during cystogenesis. Among the numerous PTMs involved, the effect of ubiquitination and deubiquitination remains largely unknown. Herein, we identified that USP28, a deubiquitinase aberrantly upregulated in patients with ADPKD, selectively removed K48-linked polyubiquitination and reversed protein degradation of signal transducer and activator of transcription 3 (STAT3). We also observed that USP28 could directly interact with and stabilize c-Myc, a transcriptional target of STAT3. Both processes synergistically enhanced renal cystogenesis. Furthermore, pharmacological inhibition of USP28 attenuated the cyst formation both in vivo and in vitro. Collectively, USP28 regulates STAT3 turnover and its transcriptional target c-Myc in ADPKD. USP28 inhibition could be a novel therapeutic strategy against ADPKD.

Abstract C065: Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer

Abstract Background: Unfortunately, the impact of immunotherapy in pancreatic cancer has, to date, been limited. Nonetheless, despite lack of success with checkpoint blockade alone, there is hope that combinations of agents may unlock the potential for immunotherapy success in pancreatic cancer. FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase that is elevated in human Pancreatic Ductal Adenocarcinoma (PDAC) tissue and correlates with high levels of fibrosis and poor CD8 cytotoxic T cell infiltration. We, and others, have already demonstrated a role for FAK in promoting tumor evasion by inducing an immunosuppressive microenvironment, specifically by regulation of cytokines such as CCL5. This has led to trials of the FAK inhibitor (defactinib) in conjunction with immunotherapy. Here we set out to improve our understanding of the role of FAK in regulating chemokine/cytokine mediated signaling in PDAC with a view to identify clinically targetable pathways with potential therapeutic benefit to pancreatic cancer patients. Methods: We combined CRISPR, NanoString, Forward Phase Protein Arrays (FPPA) and ELISA with in vivo experiments using a transplantable KrasG12Dp53R172H mouse model of Pancreatic Cancer and validated findings using human patient-derived PDAC cell lines. Results: We used CRISPR-Cas9 gene editing to deplete FAK gene expression in Panc47 cells, a syngeneic cell line isolated from PDAC arising in fully back-crossed C57BL/6 KPC mice, and reconstituted wild-type FAK (FAK-WT) expression into a clonal Panc47 FAK null (FAK-/-) cell line. Subsequent NanoString and FPPA analyses of secreted cytokines in Panc47 FAK-WT and Panc47 FAK-/- cell lines identified CXCL16 as one of the most significantly increased chemokines upon FAK depletion. Similar results were observed when CRISPR mediated FAK depletion was performed in patient-derived PDAC cell lines. Previous work in our lab has shown that depletion of Signal Transducer and Activator of Transcription-3 (STAT3), a key mediator of immune evasion, in combination with FAK depletion in orthotopically implanted PDAC tumors significantly increases CD8 T-cell infiltration into murine tumors when compared with FAK-/- tumors alone. Here we identify that dual FAK/STAT3 depletion further elevates CXCL16 expression and show that CXCR6, the receptor for CXCL16, is associated with both murine and human CD8 T-cells. Conclusions: These findings identify a FAK-dependent CXCL16:CXCR6 paracrine signaling axis associated with elevated recruitment of CD8 T-cells into PDAC tumors. This requires the dual targeting of FAK and STAT3, identifying a novel therapeutic strategy that warrants further investigation. Citation Format: Jane L. Parry, Marta Canel, Alan Serrels. Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C065.

Selectively Targeting STAT3 Using a Small Molecule Inhibitor is a Potential Therapeutic Strategy for Pancreatic Cancer.

Pancreatic cancer is the worst prognosis among all human cancers, and novel effective treatments are urgently needed. Signal transducer and activator of transcription 3 (STAT3) has been demonstrated as a promising target for pancreatic cancer. Meanwhile, selectively targeted STAT3 with small molecule remains been challenging. To specifically identify STAT3 inhibitors, more than 1.3 million compounds were screened by structure-based virtual screening and confirmed with the direct binding assay. The amino acid residues that WB436B bound to were verified by induced-fit molecular docking simulation, RosettaLigand computations, and site-directed mutagenesis. On-target effects of WB436B were examined by microscale thermophoresis, surface plasmon resonance, in vitro kinase assay, RNA sequencing, and selective cell growth inhibition assessment. In vivo studies were performed in four animal models to evaluate effects of WB436B on tumor growth and metastasis. Kaplan-Meier analyses were used to assess survival. WB436B selectively bound to STAT3 over other STAT families protein, and in vitro antitumor activities were improved by 10 to 1,000 fold than the representative STAT3 inhibitors. WB436B selectively inhibits STAT3-Tyr705 phosphorylation, STAT3 target gene expression, and the viability of STAT3-dependent pancreatic cancer cells. WB436B significantly suppresses tumor growth and metastasis in vivo and prolongs survival of tumor-bearing mice. Mechanistic studies showed that WB436B have unique binding sites located in STAT3 Src homology 2 domain. Our work presents the first-in-class selective STAT3 inhibitor WB436B as a potential therapeutic candidate for the treatment of pancreatic cancer.

The STAT3 inhibitor GPB730 enhances the sensitivity to enzalutamide in prostate cancer cells

Enzalutamide is a second-generation anti-androgen which has shown increased survival in patients with metastatic prostate cancer. However, some patients do not respond to this therapy or will develop resistance to treatment over time. Signal Transducer and Activator of Transcription 3 (STAT3) is known to be involved in castration-resistant prostate cancer and to interact with androgen receptor (AR)-signaling. This study aims to investigate the combination enzalutamide and the small molecule STAT3 inhibitor GPB730 for enhanced therapeutic effect in advanced prostate cancer in vitro. The prostate cancer cell lines LNCaP (androgen dependent) and C4–2 (androgen insensitive) were used. The effect of enzalutamide and GPB730, alone and in combination, was investigated on viability and IC50 values calculated. Enzalutamide and GPB730 treated LNCaP and C4–2 cells were subjected to western blot and QPCR analyses in order to investigate the expression of AR, STAT3 and down-stream targets. C4–2 were less sensitive to growth inhibition by enzalutamide than LNCaP cells. GPB730 enhanced the growth inhibitory effect of enzalutamide in LNCaP and C4–2 cells. The addition of GPB730 to enzalutamide decreased the IC50 values for enzalutamide by 3.3-fold for LNCaP and by 12-fold for C4–2. In C4–2 cells, GPB730 alone decreased PSA expression and enhanced the enzalutamide induced decrease in NKX3.1 expression. GPB730 and enzalutamide in combination enhanced inhibition of c-myc and survivin expression. This study suggests that enzalutamide may be combined with the STAT3 inhibitor GPB730 in order to enhance the efficacy of enzalutamide, offering a new therapeutic approach in advanced prostate cancer.

Discovery of 4-methoxy-N-(1-naphthyl)benzenesulfonamide derivatives as small molecule dual-target inhibitors of tubulin and signal transducer and activator of transcription 3 (STAT3) based on ABT-751

Overexpressed tubulin and continuously activated STAT3 play important roles in the development of many cancers and are potential therapeutic targets. A series of 4-methoxy-N -(1-naphthalene) benzenesulfonamide derivatives were designed and optimized based on β-tubulin inhibitor ABT-751 to verify whether STAT3 and tubulin dual target inhibitors have better antitumor effects. Compound DL14 showed strong inhibitory activity against A549, MDA-MB-231 and HCT-116 cells in vitro with IC50 values of 1.35 μM, 2.85 μM and 3.04 μM, respectively. Further experiments showed that DL14 not only competitively bound to colchicine binding site to inhibit tubulin polymerization with IC50 values 0.83 μM, but also directly bound to STAT3 protein to inhibit STAT3 phosphorylation with IC50 value of 6.84 μM. Three other compounds (TG03, DL15, and DL16) also inhibit this phosphorylation. In terms of single target inhibition, DL14 is slightly inferior to positive drugs, but it shows a good anti-tumor effect in vivo, and can inhibit >80% of xenograft tumor growth. This study describes a novel 4-methoxy-N-(1-naphthyl) benzenesulfonamide skeleton as an effective double-targeted anticancer agent targeting STAT3 and tubulin.

The Root Extract of Peucedanum praeruptorum Dunn Exerts Anticancer Effects in Human Non-Small-Cell Lung Cancer Cells with Different EGFR Mutation Statuses by Suppressing MET Activity.

The aim of this study was to investigate the anticancer effects of the root extract of Peucedanum praeruptorum Dunn (EPP) in human non-small-cell lung cancer (NSCLC) cells and explore the mechanisms of action. We used four types of human lung cancer cell lines, including H1299 (epidermal growth factor receptor (EGFR) wild-type), PC9 (EGFR Glu746-Ala750 deletion mutation in exon 19; EGFR tyrosine kinase inhibitor (TKI)-sensitive), H1975 (EGFR L858R/T790M double-mutant; EGFR TKI-resistant), and PC9/ER (erlotinib-resistant) cells. EPP suppressed cell growth and the colony formation of NSCLC cells in a concentration-dependent manner. EPP stimulated chromatin condensation, increased the percentage of sub-G1 phase cells, and enhanced the proportion of annexin V-positive cells, demonstrating that EPP triggered apoptosis in NSCLC cells regardless of the EGFR mutation and EGFR TKI resistance status. The phosphorylation level of the signal transducer and activator of transcription 3 (STAT3) and AKT was decreased by EPP. The expression of STAT3 target genes was also downregulated by EPP. EPP reversed hepatocyte growth factor (HGF)-induced MET phosphorylation and gefitinib resistance. Taken together, our results demonstrate that EPP exerted anticancer effects not only in EGFR TKI-sensitive NSCLC cells, but also in EGFR TKI-resistant NSCLC cells, by suppressing MET activity.

Biological Hallmarks and Emerging Strategies to Target STAT3 Signaling in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy, characterized by an abnormal accumulation of plasma cells in the bone marrow. Signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic transcription factor that modulates the transcription of multiple genes to regulate various principal biological functions, for example, cell proliferation and survival, stemness, inflammation and immune responses. Aberrant STAT3 activation has been identified as a key driver of tumorigenesis in many types of cancers, including MM. Herein, we summarize the current evidence for the role of STAT3 in affecting cancer hallmark traits by: (1) sustaining MM cell survival and proliferation, (2) regulating tumor microenvironment, (3) inducing immunosuppression. We also provide an update of different strategies for targeting STAT3 in MM with special emphasis on JAK inhibitors that are currently undergoing clinical trials. Finally, we discuss the challenges and future direction of understanding STAT3 signaling in MM biology and the clinical development of STAT3 inhibitors.

Selected Articles from This Issue

Despite previous assumptions that constitutively high Wnt signaling promotes colorectal cancer in each stage of disease, recent studies have demonstrated that reduced Wnt signaling is associated with colorectal cancer invasiveness and poor prognoses. However, little work has been done to elucidate how diminished Wnt signaling underlies aggressive colorectal cancer phenotypes. Chen and colleagues used genetic manipulation strategies and orthotopic mouse models to show that abrogating Wnt signaling increases colorectal cancer invasiveness and decreases immune responsiveness in vivo. Disrupting Wnt signaling by targeting Wnt coreceptor LDL receptor-related protein 6 (LRP6) with CRISPR/Cas9 or transducing a dominant negative version of Wnt transcriptional effector lymphoid enhancer binding factor 1 (LEF1) increases tumor submucosal invasion and dissemination in mice. Using RNA-Seq, the authors revealed that reducing Wnt signaling elevates expression of markers associated with stemness and a mesenchymal phenotype, while repressing expression of immune-stimulatory molecules and collagens. The authors used TCGA data to demonstrate that the low Wnt signaling expression profile corresponds with poor survival in colorectal cancer patients, underscoring the potential clinical relevance of dampened Wnt signaling in colorectal cancer progression.Cholangiocarcinoma (CCA) is an aggressive biliary tract cancer for which effective therapeutics are lacking. Prostaglandin E2 (PGE2)-mediated inflammation is a critical component of CCA carcinogenesis and progression. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) chemically modifies and inactivates PGE2, but its expression is often diminished in CCA. In their work, Zhang and colleagues leveraged public data to show that expression of histone methyltransferase G9a negatively correlates with 15-PGDH expression. The authors validated the G9a-15-PGDH relationship in a mouse model of CCA in which the Notch intracellular domain and constitutively active Akt are expressed via hydrodynamic injection of sleeping beauty transposase-based plasmids. shRNA-mediated G9a silencing increases 15-PGDH expression in CCA cell lines, limiting cell proliferation in vitro and tumor growth in vivo. ChIP assays demonstrated that G9a is recruited to the 15-PGDH promoter by the Myc/Max/E-box complex, which prevents signal transducer and activator of transcription 4 (STAT4) from binding the promoter and inducing 15-PGDH expression. The work presented elucidates a previously unknown mechanism underlying CCA development and progression and reveals a novel potential target for CCA therapeutic strategies.Neurofibromatosis type 2 is characterized by growth of benign nervous system tumors, such as schwannomas, which can lead to deafness, brain stem compression, and death. Neurofibromatosis type 2 is caused by mutations in tumor suppressor gene NF2. Previously, Cho and colleagues demonstrated that NF2 loss promotes elevated TβR1 signaling, resulting in phosphorylation and degradation of tumor suppressor Raf kinase inhibitor protein (RKIP). While TGF-β inhibitors halt schwannoma growth, they are not ideal therapeutics given schwannomas occur around adolescence and TGF-β is required for development. Here, Cho and colleagues developed a novel TβR1 inhibitor, Nf18001, that abrogates TβR1-mediated RKIP degradation while maintaining canonical TGF-β signaling. The authors used human and mouse schwannoma cell lines to demonstrate that Nf18001 disrupts TβR1-RKIP interactions to sustain RKIP expression and lower NF2-mutant cell viability while not influencing normal fibroblast survival. Nf18001 arrests cell cycle progression in schwannoma cells, and augments Schwann cell differentiation marker expression while diminishing expression of markers associated with cell stemness. Nf18001 also displayed efficacy in a schwannoma allograft mouse model, where treatment slowed tumor growth.The pro-tumorigenic role of signal transducer and activator of transcription 3 (STAT3) in multiple myeloma has been established, but therapeutically targeting STAT3 has proven challenging. Similarly, given its canonical role in DNA damage repair, checkpoint kinase 1 (Chk1) inhibition has been tested in anti-cancer therapeutic regimens, but with ambiguous results. In their study, Zhou and colleagues employed a gene expression array to demonstrate that Chk1 inhibition diminishes expression of oncogenic STAT3 target genes in multiple myeloma cells, suggesting Chk1 inhibition may also effectively inhibit STAT3 activity. Subsequent work showed that Chk1 directly binds to STAT3 and phosphorylates it at tyrosine 705, enabling STAT3 dimerization and DNA binding. Chk1 inhibition — pharmacologically and via shRNA silencing — diminishes STAT3 tyrosine 705 phosphorylation and inhibits STAT3 DNA binding, reducing expression of STAT3 transcriptional targets. Chk1 inhibition abrogates STAT3 tyrosine 705 phosphorylation in IL-6–dependent, -independent, and bortezomib-resistant cells, as well as in patient primary myeloma mononuclear cells and multiple myeloma cells implanted into mice, suggesting Chk1 inhibition may provide therapeutic efficacy against multiple myeloma through a STAT3-related mechanism.

STAT1 and STAT3 Exhibit a Crosstalk and Are Associated with Increased Inflammation in Hepatocellular Carcinoma.

Simple SummaryLiver cancer is the fourth-leading cause of cancer-related mortality worldwide and lacks effective therapies. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common types of liver cancer and both are associated with underlying inflammatory diseases. Thereby, interleukin-6 (IL-6)-mediated STAT3 signaling is critically involved in early carcinogenesis and disease progression. Here, we assessed the interplay between STAT1 and STAT3 in IL-6 signaling in vitro and studied the activation of STAT1 and STAT3 in a cohort of 124 HCC and a cohort of 138 CCA patients by immunohistochemistry. We found that IL-6 induced STAT1 transcriptional activity upon STAT3 depletion, suggesting that HCC tumor cells may activate both STAT1 and STAT3 signaling under pro-inflammatory conditions. Furthermore, HCC patient tissues showed a strong positive correlation of STAT1 and STAT3 activation in distinct patient groups. These patients also exhibited a high degree of immune cell infiltration, suggesting that these tumors are immune “hot”.Liver cancers, which are mostly hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are very aggressive tumors with poor prognosis. Therapeutic options with curative intent are largely limited to surgery and available systemic therapies show limited benefit. Signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) are key transcription factors activated by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6 (IL-6). In this study, we combined in vitro cell culture experiments and immunohistochemical analyses of human HCC (N = 124) and CCA (N = 138) specimens. We observed that in the absence of STAT3, IL-6 induced the activation of STAT1 and its target genes suggesting that IL-6 derived from the tumor microenvironment may activate both STAT1 and STAT3 target genes in HCC tumor cells. In addition, STAT1 and STAT3 were highly activated in a subset of HCC, which exhibited a high degree of infiltrating CD8- and FOXP3-positive immune cells and PD-L1 expression. Our results demonstrate that STAT1 and STAT3 are expressed and activated in HCC and tumor infiltrating immune cells. In addition, HCC cases with high STAT1 and STAT3 expression also exhibited a high degree of immune cell infiltration, suggesting increased immunological tolerance.

Adipose-Derived Mesenchymal Stem Cell-Derived Exosomal miR-301a-3p Regulates Airway Smooth Muscle Cells During Asthma by Targeting STAT3.

BackgroundAsthma is a chronic inflammatory disease featured by inflammation and remodeling of airway. Adipose-derived mesenchymal stem cell (ADSCs)-derived exosomal miRNAs have been suggested as promising therapeutic manners for diseases.MethodsADSCs and airway smooth muscle cells (ASMCs) were isolated from SD rats. Flow cytometry was conducted to detect the surface biomarkers of isolated cells. Exosomes were extracted by sequentially centrifuge method and identified by Western blotting and nanoparticle tracking analysis (NTA). Uptake of exosomes by ASMCs was detected by confocal assay. ASMCs were treated with platelet-derived growth factor-BB (PDGF-BB) to mimic cell remodeling and inflammation. Cell counting 8 (CCK-8), Transwell, and flow cytometry were performed to determine the viability, migration, and apoptosis of ASMCs. Release of inflammatory factors was detected by enzyme-linked immunosorbent assay (ELISA). Levels of RNAs and proteins were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. Interaction between miR-301a-3p and signal transducer and activator of transcription 3 (STAT3) was determined by luciferase reporter gene assay. The effect of Exosomal miR-301a-3p was analyzed in ovalbumin (OVA)-induced asthma mouse model.ResultsADSCs-derived exosomes could be effectively internalized by ASMCs. Exosomal miR-301a-3p notably suppressed the PDGF-BB-stimulated proliferation and migration of ASMCs, and enhanced apoptosis, as well as decreased the secretion of inflammatory factors. MiR-301a-3p directly targeted the 3ʹUTR region of STAT3. STAT3 overexpression reversed the suppressive effects of exosomal miR-301a-3p on ASMCs under PDGF-BB stimulation. The expression of miR-301a-3p and STAT3 was negative correlation in specimen from patients with asthma. Exosomal miR-301a-3p inhibited OVA-induced lung injury by targeting STAT3 in mice.ConclusionThis study exposed that exosomal miR-301a-3p from ADSCs could effectively alleviate PDGF-BB-stimulated remodeling and inflammation of ASMCs via targeting STAT3, presented ADSCs-derived exosomal miR-301a-3p as a promising therapeutic approach for asthma.

18[Formula: see text]-Glycyrrhetinic Acid Inhibits TGF-[Formula: see text]-Induced Epithelial-to-Mesenchymal Transition and Metastasis of Hepatocellular Carcinoma by Targeting STAT3.

18[Formula: see text]-glycyrrhetinic acid (GA) is the active ingredient of the traditional Chinese medicinal herb Glycyrrhizae radix et rhizoma. We previously demonstrated that GA inhibited tumor growth in hepatocellular carcinoma (HCC). However, the effect of GA on transforming growth factor-[Formula: see text] (TGF-[Formula: see text]-induced epithelial-mesenchymal transition (EMT) and metastasis were still unclear. In this study, in vitro transwell assays and immunofluorescence (IF) demonstrated that GA inhibited TGF-[Formula: see text]-induced migration, invasion and EMT of HCC cells. However, it had little effect on the inhibition of proliferation by TGF-[Formula: see text]. Moreover, we confirmed that GA suppressed the metastasis of HCC cells in vivousing an ectopic lung metastasis model. Furthermore, we found that GA inhibited TGF-[Formula: see text]-induced EMT mainly by reducing the phosphorylation of signal transducer and activator of transcription 3 (STAT3), which played an essential role in TGF-[Formula: see text]-induced EMT and cell mobility. Mechanistically, GA inhibited the phosphorylation of STAT3 by increasing the expression of Src homology 2 domain-containing protein tyrosine phosphatases 1 and 2 (SHP1 and SHP2). Therefore, we concluded that GA inhibited TGF-[Formula: see text]-induced EMT and metastasis via the SHP1&SHP2/STAT3/Snail pathway. Our data provide an attractive therapeutic target for future multimodal management of HCC.

Targeting STAT3 prevents bile reflux-induced oncogenic molecular events linked to hypopharyngeal carcinogenesis.

The signal transducer and activator of transcription 3 (STAT3) oncogene is a transcription factor with a central role in head and neck cancer. Hypopharyngeal cells (HCs) exposed to acidic bile present aberrant activation of STAT3, possibly contributing to its oncogenic effect. We hypothesized that STAT3 contributes substantially to the bile reflux‐induced molecular oncogenic profile, which can be suppressed by STAT3 silencing or pharmacological inhibition. To explore our hypothesis, we targeted the STAT3 pathway, by knocking down STAT3 (STAT3 siRNA), and inhibiting STAT3 phosphorylation (Nifuroxazide) or dimerization (SI3‐201; STA‐21), in acidic bile (pH 4.0)‐exposed human HCs. Immunofluorescence, luciferase assay, Western blot, enzyme‐linked immunosorbent assay and qPCR analyses revealed that STAT3 knockdown or pharmacologic inhibition significantly suppressed acidic bile‐induced STAT3 activation and its transcriptional activity, Bcl‐2 overexpression, transcriptional activation of IL6, TNF‐α, BCL2, EGFR, STAT3, RELA(p65), REL and WNT5A, and cell survival. Our novel findings document the important role of STAT3 in bile reflux‐related molecular oncogenic events, which can be dramatically prevented by STAT3 silencing. STA‐21, SI3‐201 or Nifuroxazide effectively inhibited STAT3 and cancer‐related inflammatory phenotype, encouraging their single or combined application in preventive or therapeutic strategies of bile reflux‐related hypopharyngeal carcinogenesis.

Ginsenoside Rg1 Inhibits Microglia Pyroptosis Induced by Lipopolysaccharide Through Regulating STAT3 Signaling.

PurposeNeuroinflammation runs through the whole process of nervous system diseases and brain injury. Inflammasomes are thought to be especially relevant to immune homeostasis, and their dysregulation contributes to pyroptosis. The natural compound Ginsenoside Rg1 has been shown to possess anti-inflammatory effects; however, its underlying mechanisms are not entirely clear. Therefore, this study was undertaken to investigate the role and mechanisms of Rg1 in regulating the production of inflammasomes and pyroptosis of microglia in vivo and in vitro.MethodsBV-2 microglial cells were pretreated with Rg1, stattic and interleukin-6 (IL-6), and then stimulated with lipopolysaccharide (LPS) (2μg/mL). Hoechst staining and Annexin V-FITC/PI assay were then carried out. The expression levels of cleaved-caspase-1, pro-caspase-1, interleukin-1β (IL-1β), mature-IL-1β, gasdermin D (GSDMD), activated NH(2)-terminal fragment of GSDMD (GSDMD-N), NOD-, LRR- and pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), absent in melanoma 2 (AIM2), signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 in BV-2 were detected by Western blotting. Additionally, immunofluorescence staining was used to determine the expression of NLRP3 and p-STAT3 in postnatal rat brain and BV-2 microglia subjected to LPS stimulation and Rg1 pretreatment. The targets of transcription factor STAT3 were predicted by hTFtarget and chromatin immunoprecipitation (ChIP) was used to confirm the interaction between STAT3 and AIM2.ResultsWe showed here that Rg1 effectively inhibited the expression of inflammasomes and microglia pyroptosis induced by LPS. The targets predicted data of Rg1 from Swiss target prediction database showed STAT3 had the highest thresholds of probability score. Rg1 can regulate the phosphorylation of STAT3, which binds to the promoter region of inflammasome AIM2.ConclusionIt is suggested that STAT3 signaling can initiate the transcription activity of AIM2. Rg1 regulates microglia pyroptosis in neuroinflammation induced by LPS through targeting STAT3 signaling.

A Phase 1 Study of the Combination of Acalabrutinib and AZD9150 in Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma

A Phase 1 Study of the Combination of Acalabrutinib and AZD9150 in Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Acacetin Inhibits the Growth of STAT3-Activated DU145 Prostate Cancer Cells by Directly Binding to Signal Transducer and Activator of Transcription 3 (STAT3)

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in the formation and growth of human cancer. Therefore, STAT3 is a therapeutic target for cancer drug discovery. Acacetin, a flavone present in various plants, inhibits constitutive and inducible STAT3 activation in STAT3-activated DU145 prostate cancer cells. Acacetin inhibits STAT3 activity by directly binding to STAT3, which we confirmed by a pull-down assay with a biotinylated compound and two level-free methods, namely, a drug affinity responsive target stability (DARTS) experiment and a cellular thermal shift assay (CETSA). Acacetin inhibits STAT3 phosphorylation at the tyrosine 705 residue and nuclear translocation in DU145 cells, which leads to the downregulation of STAT3 target genes. Acacetin then induces apoptosis in a time-dependent manner. Interestingly, acacetin induces the production of reactive oxygen species (ROS) that are not involved in the acacetin-induced inhibition of STAT3 activation because the suppressed p-STAT3 level is not rescued by treatment with GSH or NAC, which are general ROS inhibitors. We also found that acacetin inhibits tumor growth in xenografted nude mice. These results suggest that acacetin, as a STAT3 inhibitor, could be a possible drug candidate for targeting STAT3 for the treatment of cancer in humans.

Inhibition of STAT3/PD-L1 and Activation of miR193a-5p Are Critically Involved in Apoptotic Effect of Compound K in Prostate Cancer Cells.

Since the signal transducer and activator of transcription 3 (STAT3)/programmed death-ligand 1 (PD-L1) signaling plays an important role in tumor-immune microenvironments, in the present study, the role of STAT3/PD-L1 signaling in the apoptotic mechanism of an active ginseng saponin metabolite compound K (CK) was investigated in human prostate cancer cells. Here, CK exerted significant cytotoxicity without hurting RWPE1 normal prostate epithelial cells, increased sub-G1 and cleavage of Poly ADP-ribose polymerase (PARP) and attenuated the expression of pro-PARP and Pro-cysteine aspartyl-specific protease3 (pro-caspase-3) in LANCap, PC-3 and DU145 cells. Further, CK attenuated the expression of p-STAT3 and PD-L1 in DU145 cells along with disrupted the binding of STAT3 to PD-L1. Furthermore, CK effectively abrogated the expression of p-STAT3 and PD-L1 in interferon-gamma (INF-γ)-stimulated DU145cells. Additionally, CK suppressed the expression of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), interleukin 6 (IL-6) and interleukin 10 (IL-10) as immune escape-related genes in DU145 cells. Likewise, as STAT3 targets genes, the expression of CyclinD1, c-Myc and B-cell lymphoma-extra-large (Bcl-xL) was attenuated in CK-treated DU145 cells. Notably, CK upregulated the expression of microRNA193a-5p (miR193a-5p) in DU145 cells. Consistently, miR193a-5p mimic suppressed p-STAT3, PD-L1 and pro-PARP, while miR193a-5p inhibitor reversed the ability of CK to attenuate the expression of p-STAT3, PD-L1 and pro-PARP in DU145 cells. Taken together, these findings support evidence that CK induces apoptosis via the activation of miR193a-5p and inhibition of PD-L1 and STAT3 signaling in prostate cancer cells.

Targeting STAT3 Signaling Pathway in Colorectal Cancer.

Signal transducer and activator of transcription 3 (STAT3) is a critical transcription factor that has been firmly associated with colorectal cancer (CRC) initiation and development. STAT3 mediates key inflammatory mechanisms in colitis-associated cancer, becomes excessively activated in CRC, and enhances cancer cell proliferation, tumor growth, angiogenesis, invasion, and migration. STAT3 hyperactivation in malignant cells, surrounding immune cells and cancer-associated fibroblasts, mediates inhibition of the innate and adaptive immunity of the tumor microenvironment, and, therefore, tumor evasion from the immune system. These features highlight STAT3 as a promising therapeutic target; however, the mechanisms underlying these features have not been fully elucidated yet and STAT3 inhibitors have not reached the clinic in everyday practice. In the present article, we review the STAT3 signaling network in CRC and highlight the current notion for the design of STAT3-focused treatment approaches. We also discuss recent breakthroughs in combination immunotherapy regimens containing STAT3 inhibitors, therefore providing a new perception for the clinical application of STAT3 in CRC.