STAT3 Knockdown Research Articles

Cancer-associated fibroblasts promote the stemness of CD24+ liver cells via paracrine signaling.

Cancer stem cells (CSCs), which support tumor progress in hepatocellular carcinoma (HCC) developed in fibrotic or cirrhotic livers, are regulated by the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are the major component of the tumor stroma in HCC; however, the mechanisms by which CAFs contribute to stemness maintenance remain largely unknown. Here, we found that the expression of CD24 was high in HCC tissues compared with adjacent normal liver tissues, and positively correlated with the poor prognosis and α-SMA expression in CAFs. CD24+ cells isolated from HCC cell lines exhibited stemness properties of self-renewal, chemotherapy resistance, metastasis, and tumorigenicity in NOD/SCID mice. Moreover, CAF-derived HGF and IL6 enhanced the stemness properties of CD24+ cells via activating STAT3 Tyr705 phosphorylation. Blockade of HGF/c-Met or IL6/IL6R signaling significantly abolished the effect of CAFs on stemness properties, which compromised the activation of STAT3 pathway in CD24+ cells. Meanwhile, knockdown of STAT3 in CD24+ cells notably attenuated CAF-induced stemness characteristics of CD24+ cells. Furthermore, in HCC patients, higher expression of phospho-STAT3 was also demonstrated to be positively correlated with poor clinical outcomes. In summary, HGF and IL6 secreted by CAFs promoted the stemness properties of CD24+ cells through the phosphorylation of STAT3 signaling, and targeting the paracrine pathways may provide a new therapeutic strategy for HCC. KEY MESSAGES: CD24, identified as a marker for HCC CSCs, was positively correlated with the poor prognosis and α-SMA expression in CAFs. CAFs promoted self-renewal, chemotherapy resistance, metastasis, and tumorigenicity of CD24+ HCC cells. HGF and IL6 secreted by CAFs promoted the stemness properties of CD24+ HCC cells through the phosphorylation of STAT3.

Ambient fine particulate matter (PM2.5) induces oxidative stress and pro-inflammatory response via up-regulating the expression of CYP1A1/1B1 in human bronchial epithelial cells in vitro

We investigated the mechanism responsible for the oxidative stress and pro-inflammatory response triggered by PM2.5 collected from Nanjing of China. Two human bronchial epithelia cell lines (HBE and BEAS-2B) were used. Human gene expression profile microarray was performed to investigate the alteration of gene expression in PM2.5-treated HBE cells. The results of ROS assay and ELISA indicated that PM2.5 (150 μg/ml) increased the level of cellular reactive oxygen species (ROS) and promoted the release of interleukin-6 (IL-6) in HBE cells. CYP1A1 and CYP1B1 were the top two up-regulated genes by PM2.5 (150 μg/ml, 48 h of exposure) in HBE cells. Co-knockdown of CYP1A1/1B1 by siRNA substantially inhibited PM2.5-induced ROS generation, IL-6/IL-8 secretion and STAT3/P-STAT3 expression. Similarly, the knockdown of STAT3 also effectively inhibited PM2.5-induced rise in ROS level and IL-6/IL-8 secretion. In summary, PM2.5 mediated oxidative stress and pro-inflammatory response via up-regulating the expression of CYP1A1/1B1 in two human bronchial epithelial cell lines.

Interplay of transcription factors STAT3, STAT1 and AP-1 mediates activity of the matrix metallo-proteinase-1 promoter in colorectal carcinoma cells.

Signal Transducers (STATs) 1 and 3 and Activator Protein 1 (AP-1) are transcription factors involved in the development of malignancy in colorectal carcinoma (CRC). Matrix Metalloproteinase 1 (MMP-1) is a protease frequently dysregulated in de-differentiated and invasive cancer cells. Its expression is influenced by STAT and AP-1 transcription factors. We studied their contributions to transcriptional regulation of MMP-1 in colorectal carcinoma (CRC) cells. Both STAT3 and AP-1 contribute individual expression-inducing and additive effects and interact with the MMP-1 promoter. DNA binding of AP-1 protein c-Jun is stimulation-independent but modulated by STAT3 and a STAT recognition DNA element. Activated STAT3 showed a suppressive effect on AP-1-mediated MMP-1 mRNA upregulation as shown by STAT3 knockdown. Surprisingly, activated STAT1 overcame STAT3-dependent repression of AP-1-driven MMP-1 expression. Moreover, combined STAT3, STAT1 and AP-1 activities evoked maximal MMP-1 mRNA levels in a synergistic manner. Our results suggest a dominant role of AP-1 in transcriptional upregulation of MMP-1 in CRC cells which is modulated by joint functions of STAT3 and STAT1. The individual and combinatorial activity of these factors is of diagnostic and prognostic interest.

MicroRNA-221 sensitizes chronic myeloid leukemia cells to imatinib by targeting STAT5

MicroRNAs (miRNAs) are involved in various processes from the development to drug resistance of tumors, including chronic myeloid leukemia (CML). In this study, we examined the STAT5-related miRNA-expression profile in CML cell lines (K562 and imatinib-resistant K562/G) by quantitative real-time reverse-transcriptase polymerase chain reactions. MiR-221 expression was markedly decreased in K562/G cells and peripheral blood mononuclear cells from patients with treatment failure, when compared to imatinib-sensitive CML cells and patients with optimal responses respectively. We also observed the expression of STAT5 inversely correlated with miR-221 expression in K562 and KBM5 cells. Additionally, STAT5 was validated as a direct target of miR-221 in dual-luciferase reporter vector assays. MiR-221 restoration and STAT5 knockdown in K562/G cells increased the sensitivity of CML cells to imatinib by reducing the Bcl2: Bax ratio. Collectively, our data suggested that miR-221–STAT5 axis played crucial roles in controlling the sensitivity of CML cells to imatinib.

PPARα-independent action against metabolic syndrome development by fibrates is mediated by inhibition of STAT3 signalling.

Metabolic syndrome (MS) is the concurrence of at least three of five medical conditions: obesity, high blood pressure, insulin resistance, high serum triglyceride (TG) and low serum high-density lipoprotein levels. While fibrates are used to treat disorders other than the lowering serum TG, the mechanism by which fibrates decrease MS has not been established. In this study, wild-type and Ppara-null mice fed a medium-fat diet (MFD) were administered gemfibrozil and fenofibrate for 3 months respectively, to explore the effect and action mechanism. In Ppara-null mice, MFD treatment increased body weight, adipose tissue, serum TG and impaired glucose tolerance. These phenotypes were attenuated in two groups treated with gemfibrozil and fenofibrate. The STAT3 pathway was activated in adipose and hepatic tissues in positive control, and inhibited in groups treated with gemfibrozil and fenofibrate. The above phenotypes and inflammation were not observed in any wild-type group. In 3T3-L1 adipogenic stem cells treated with high glucose, STAT3 knockdown greatly decreased the number of lipid droplets. Low dose of clinical fibrates was effective against MS development independent of PPARα, and this action was mediated by STAT3 signalling inhibition in adipose tissue and, to a lesser extent, in hepatic tissues.

Highlights of This Issue

Emerging technologies bring the promise of enhancing cancer detection and providing novel methods for monitoring tumor response. Liquid biopsy is one such emerging platform that allows tumor monitoring without the need for surgical interventions. The importance of liquid biopsy is more eminent when applied to surgically nonresectable tumors. Panditharatna and colleagues designed and developed a sensitive and specific assay for probing tumor-associated mutant DNA in liquid biopsies obtained from children diagnosed with brain cancers. The significance of this work is highlighted in its ability to correlate liquid biopsy data with MR imaging and provide its feasibility in the context of clinical trials.The heterogeneity of pancreatic ductal adenocarcinoma (PDAC) is well-recognized molecularly and reflected clinically in variable disease presentations and treatment responses. Interrogating that biological heterogeneity for patients has been challenging, partly related to obtaining sufficient tumor samples. Koay and colleagues addressed this challenge by developing a classification of PDAC morphology on standard-of-care computed tomography (CT) scans, which are ubiquitous in clinics. They discovered two distinct imaging phenotypes that correlated with genetic aberrations, stromal content, and clinical outcomes. Thus, a CT-based biomarker could be used to separate PDAC into distinct subtypes, with implications for treatment selection, response evaluation, and early detection.Prostate cancers show remarkable resistance to emerging immunotherapies, partly due to tolerogenic STAT3 signaling in tumor-associated myeloid cells. Moreira and colleagues developed an oligonucleotide strategy combining STAT3 knock-down with Toll-like Receptor-9 (TLR9) immune-stimulation in the microenvironment of bone-localized prostate tumors. Systemic administration of these oligonucleotides triggered immune-mediated eradication of two genetically different prostate tumor models in mice, which relied on both innate and adaptive CD8/CD4 T-cell–mediated immunity as verified immune cell depletion studies. The bifunctional oligonucleotide design, both immunostimulatory and tolerance-breaking, offers an opportunity for the therapy of immunologically “cold” tumors, such as prostate cancers.MET-targeted therapies are clinically effective in MET exon 14 mutant non–small cell lung cancers (NSCLC); however, resistance inevitably develops. While several MET secondary mutations imparting resistance have been reported, Bahcall and colleagues describe one of the first examples of a resistance mechanism involving alternative signaling. The study characterizes acquired amplification of wild-type (WT) KRAS as a recurrent molecular mechanism behind crizotinib resistance in three cases of MET exon 14 mutant NSCLC, using in vitro assays and patient-derived xenografts. In these models, KRAS amplification uncouples MET from downstream effectors, induces expression of EGFR ligands, and confers hypersensitivity to ligand-dependent and independent activation. The auxiliary reliance on PI3K renders these models vulnerable to dual MET/PI3K inhibition as a promising therapeutic strategy.

STAT3 Inhibition Combined with CpG Immunostimulation Activates Antitumor Immunity to Eradicate Genetically Distinct Castration-Resistant Prostate Cancers.

Prostate cancers show remarkable resistance to emerging immunotherapies, partly due to tolerogenic STAT3 signaling in tumor-associated myeloid cells. Here, we describe a novel strategy combining STAT3 inhibition with Toll-like Receptor 9 (TLR9) stimulation to unleash immune response against prostate cancers regardless of the genetic background. We developed and validated a conjugate of the STAT3 antisense oligonucleotide (ASO) tethered to immunostimulatory TLR9 agonist (CpG oligonucleotide) to improve targeting of human and mouse prostate cancer and myeloid immune cells, such as myeloid-derived suppressor cells (MDSC). CpG-STAT3ASO conjugates showed improved biodistribution and potency of STAT3 knockdown in target cells in vitro and in vivo. Systemic administration of CpG-STAT3ASO (5 mg/kg) eradicated bone-localized, Ras/Myc-driven, and Ptenpc -/- Smad4pc -/- Trp53c -/- prostate tumors in the majority of treated mice. These antitumor effects were primarily immune-mediated and correlated with an increased ratio of CD8+ to regulatory T cells and reduced pSTAT3+/PD-L1+ MDSCs. Both innate and adaptive immunity contributed to systemic antitumor responses as verified by the depletion of Gr1+ myeloid cells and CD8+ and CD4+ T cells, respectively. Importantly, only the bifunctional CpG-STAT3ASO, but not control CpG oligonucleotides, STAT3ASO alone, or the coinjection of both oligonucleotides, succeeded in recruiting neutrophils and CD8+ T cells into tumors. Thus, the concurrence of TLR9 activation with STAT3 inhibition in the same cellular compartment is indispensable for overcoming tumor immune tolerance and effective antitumor immunity against prostate cancer. The bifunctional, immunostimulatory, and tolerance-breaking design of CpG-STAT3ASO offers a blueprint for the development of effective and safer oligonucleotide strategies for treatment of immunologically "cold" human cancers.

JAK-STAT3 Pathway Regulates CD38 Expression on Multiple Myeloma Cells

CD38 is highly expressed on malignant plasma cells from patients with multiple myeloma (MM), and anti-CD38 monoclonal antibodies (i.e. daratumumab, SAR650984) trigger cytotoxicity through antibody-dependent cellular cytotoxicity (ADCC) and activation of complement-dependent cytotoxicity (CDC). Although remarkable response has been observed in relapsed and refractory MM, relapse of disease is common. To improve the clinical efficacy of CD38-targeting immunotherapies, novel combination treatment strategies have been evaluated in preclinical studies and clinical trials. For example, all-trans retinoic acid or histone deacetylase inhibitors enhance daratumumab-induced cytotoxicity via upregulation of CD38 expression. To date, however, the biologic impact of the bone marrow microenvironment on CD38-targeting immunotherapies has not been characterized. In this study, we evaluated the impact of bone marrow stromal cells (BMSCs) from MM patients on CD38 expression and anti-CD38 Antibody-induced ADCC.We first cultured MM cells (RPMI8226, MM.1S, MOLP8) with culture supernatant from BMSCs and measured CD38 expression by flow cytometry. A significant reduction of CD38 expression on all MM cell lines was noted in a time-dependent fashion. For example, CD38 expression (mean fluorescence intensity) was reduced 44%, 32%, and 42% on RPMI8226, MM.1S and MOLP8 cells, respectively, after 48 h culture with BMSC supernatants. To identify mediators of this effect, we next examined the effect of MM-relevant cytokines (IL-6, IGF-1, IL-1β) on CD38 expression. Importantly, IL-6 significantly downregulated CD38 expression on MM cells whereas IGF-1 or IL-1β had no significant impact. We further performed quantitative real-time PCR to evaluate CD38 mRNA level in RPMI8226 and MOLP8 cells cultured with BMSC supernatant or IL-6 and confirmed that CD38 transcript was downregulated by both treatments in these cells. Since IL-6, but not IGF-1, downregulated CD38, we hypothesized that JAK-STAT3 pathway might mediate this IL-6 triggered downregulation of CD38 expression. As with IL-6 treatment, transfection of MM.1S cells with constitutively active STAT3 significantly reduced CD38 expression. Conversely, knockdown of STAT3 by siRNA partially restored CD38 expression on RPMI8226 cells in the presence of IL-6. Moreover, we cultured RPMI8226 and MM.1S cells with JAK inhibitor ruxolitinib (1, 3, 10 μM) in the presence of BMSC supernatant or IL-6. We observed significant recovery of CD38 expression on MM cells. We next evaluated the impact of these modulations of CD38 expression on ADCC. Natural killer cell-resistant RPMI8226 cells were pre-treated with BMSC supernatant for 48 h and then labeled with calcein-AM for 30 min, followed by co-culture with peripheral blood mononuclear cells from healthy donors (effector/target ratio of 30:1) in the presence of anti-CD38 antibody (1 μg/ml of SAR650984). As expected, ADCC was reduced by BMSC supernatant.Our studies, therefore, show that BMSC supernatant downregulates CD38 expression through IL-6-STAT3 signaling pathway in MM cells, and thereby reduces sensitivity to CD38-targeted ADCC in MM cells. They provide the rationale for novel treatment strategies of CD38-targeting immunotherapy in combination with JAK-STAT pathway inhibitors to enhance sensitivity or overcome resistant to CD38-targeted ADCC in the context of the MM BM microenvironment. DisclosuresAnderson:Celgene: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Bristol Myers Squibb: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder.

Stat3 Regulates Alloreactive T Cell Susceptibility Towards PD-L1-Mediated Tolerance Signaling, Gvhd and GVL Activity

Prevention of graft versus host disease (GVHD) while preserving graft-versus-leukemia/lymphoma (GVL) activity of alloreactive donor T cells remains an elusive and long-sought goal. We and others reported that Stat3 deficiency in donor T cells results in expansion of Foxp3+ Treg cells and reduction of pathogenic Th17 cells, leading to prevention of chronic GVHD. We have now tested whether Stat3 deficiency in donor T cells can prevent acute GVHD while preserving GVL activity.We transplanted wild-type (WT) or Stat3-/- T cells from C57BL/6 (H-2b) donors into irradiated WT BALB/c recipients bearing B cell lymphoma line BCL1 or aggressive acute lymphocytic leukemia (ALL) cells. WT donor T cells caused acute GVHD, as expected. Stat3-/- donor T cells did not cause acute GVHD but still had strong GVL activity. This separation of GVL activity from GVHD was associated with augmented expansion and impaired tolerance by Stat3-/- T cells in lymphoid tissues such as the spleen. In contrast, tolerance by Stat3-/- T cells was enhanced in GVHD target tissues by different mechanisms. The yield of Stat3-/- T cells in gut tissues was lower than the yield of WT T cells due to the markedly higher apoptosis of Stat3-/- T cells compared to WT T cells. The yield of Stat3-/- T cells in the lung and liver was not lower than the yield of WT T cells, but expression of anergy/exhaustion markers such as PD-1 and KLRG1 was markedly higher in Stat3-/- T cells than in WT T cells.We recently reported that GVHD tissue expression of PD-L1 can tolerize donor CD8+ T cells in the absence of donor CD4+ T cells, but not when CD4+ and CD8+ T cells were given together. In contrast, PD-L1 expression in recipient GVHD target tissues tolerized Stat3-/- donor T cells when both CD4+ T cells and CD8+ T cells were given together. The induction of tolerance in Stat3-/- T cells by GVHD target tissue expression of PD-L1 depended on PD-L1/PD-1 interaction, which led to marked reduction of glycolysis in Stat3-/- T cells but not in WT T cells. These observations fit with previous findings that enhanced glycolysis was associated with enhanced GVHD activity of alloreactive T cells, as reported by Xue-zhong Yu's group. Our results indicate that Stat3 deficiency in donor T cells can augment their susceptibility to tissue PD-L1-mediated tolerance mechanisms such as down-regulation of glycolysis. Current experiments are testing whether Stat3 knockdown in mature T cells in the graft can prevent GVHD while preserving GVL activity. (Grant support: R01AI066008 to Zeng) DisclosuresNo relevant conflicts of interest to declare.

Combining Dasatinib and AC220 Reduces Stroma-Based pSTAT5Y694 in FLT3-ITD+ AML and Overcomes FLT3 TKI Resistance

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis. FLT3 internal tandem duplications (ITDs) are found in ~30% of AML patients and are associated with inferior survival. Addition of FLT3 tyrosine kinase inhibitors (TKIs) such as midostaurin improve survival of AML treated with cytotoxic chemotherapy. However, relapse rates remain unacceptably high. Protection by stroma-derived survival signals is thought to enable survival of leukemia initiating cells (LICs) in the presence of FLT3 TKIs, providing a reservoir for subsequent overt relapse. We previously reported that HS-5 conditioned medium (CM) rescued FLT3-ITD positive AML cell lines and primary cells from the effects of the 2nd generation FLT3 TKI AC220 (quizartinib) as compared to cells cultured in regular medium (RM). Earlier work from our lab and others has demonstrated that HS-5 CM increases pSTAT3Y705 in chronic myeloid leukemia cells treated with BCR-ABL1 TKIs, leading to TKI resistance, while pSTAT5Y694 levels remain under the control of the BCR-ABL1 kinase (Eiring et al, Leukemia, 2015). Similar to BCR-ABL1, FLT3 signaling results in potent pSTAT5Y694 activation. We hypothesized that STAT3 and/or STAT5 activation contributes to stroma-mediated protection of FLT3-ITD+ AML cells upon FLT3 inhibition with AC220.We found that FLT3-ITD+ cell lines and primary cells grown in HS-5 CM exhibited strong upregulation of pSTAT3Y705 and pSTAT5Y694 that was unaffected by treatment with 10nM AC220. In RM, pSTAT3Y705 was undetectable irrespective of AC220 dose and pSTAT5Y694 was abolished by 10nM AC220. shRNA knockdown experiments of STAT3 and STAT5 in FLT3-mutated cell lines grown in CM and treated with AC220 showed that STAT5 knockdown alone consistently resulted in greater inhibition of cell proliferation than STAT3 knockdown alone. This suggests that in FLT3-ITD+ AML cells cultured in CM, upregulation of pSTAT5Y694 by stroma-derived soluble factors is the main contributor to AC220 resistance.We decided to use a candidate approach to identify the upstream kinases leading to pSTAT5Y694, focusing on inhibitors of pathways previously implicated in STAT5 activation in AML. We used phosphoflow to quantify pSTAT5Y694 in MOLM-13 cells grown for 24 hours in CM in the presence of 10nM AC220 combined with ruxolitinib, dasatinib, ibrutinib, PD173074 (FGFR1i) or PRT062607 (SYKi). Results from this screen indicated that the combination of dasatinib and AC220 most effectively decreased pSTAT5Y694 in MOLM-13 cells cultured in CM (minimum dasatinib dose =100nM). To further validate dasatinib as a candidate for combination drug treatment, we performed cell proliferation experiments in MOLM-13 cells cultured in CM for 72h with graded concentrations of AC220 and the candidate inhibitors at a minimum candidate drug dose of 100nM. Of the four inhibitors tested, the combination of dasatinib and AC220 most significantly decreased the IC50 of AC220 in MOLM-13 cells grown in CM (IC50 AC220 alone: 11.09nM; IC50 AC220 + 100nM dasatinib: 0.41nM; p<0.05, n=3).It has recently been reported that FLT3-ITD AML cells harbor a highly glycolytic phenotype that can be partially suppressed by FLT3 TKIs (Huntly et al, Blood, 2018). Our preliminary Seahorse experiments show that the combination of dasatinib and AC220 more effectively inhibits glycolysis in MOLM-13 cells than AC220 alone, with differential modulation in CM versus RM. These results suggest that dasatinib inhibits a metabolic pathway critical to maintaining glycolysis in MOLM-13 cells challenged by AC220-based FLT3 inhibition. Moreover, these data indicate that extrinsic signals contribute to metabolic reprogramming of FLT3+ AML cells, enabling survival despite FLT3 inhibition. Experiments are underway to delineate how CM contributes to metabolic resistance and how dasatinib intercepts this survival signal. Our results support the concept of combining AC220 with dasatinib to reduce residual leukemia in FLT3+ AML and prolong survival. Further investigation is ongoing and will be reported at the time of abstract presentation. DisclosuresDeininger:Blueprint: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

MiR-221 Sensitizes Chronic Myeloid Leukemia Cells to Imatinib By Targeting STAT5

Imatinib, a tyrosine-kinase inhibitors (TKIs), is the first-line treatment in the early chronic phase of chronic myeloid leukemia (CML). In the last decade, the survival of patients with CML has significantly increased with the use of TKIs. However, the drug resistance is one major barrier in CML treatment. At present, although the research on CML TKIs resistance mechanism has made great progress, the underlying mechanism is complex and still not fully elucidated, especially the lack of understanding of the upstream common mechanism. MicroRNAs (miRNAs) are involved in various processes from the development to drug resistance of tumors, including chronic myeloid leukemia (CML). In this work, we examined the STAT5-related miRNA-expression profile in CML cell lines (K562 and imatinib-resistant K562/G) by quantitative real-time reverse-transcriptase polymerase chain reactions. MiR-221 expression was markedly decreased in K562/G cells and peripheral blood mononuclear cells from patients with treatment failure, when compared to K562 cells and patients with optimal responses respectively. We also observed the expression of STAT5 expression inversely correlated with miR-221 expression in CML cells. Additionally, STAT5 was validated in this study as a direct target of miR-221 in dual-luciferase reporter vector assays. MiR-221 restoration and STAT5 knockdown in K562/G cells increased the sensitivity of CML cells to imatinib by reducing the Bcl2: Bax ratio. Collectively, our data suggest that miR-221-STAT5 axis played crucial roles in controlling the sensitivity of CML cells to imatinib. DisclosuresNo relevant conflicts of interest to declare.

PCNA and JNK1-Stat3 pathways respectively promotes and inhibits diabetes-associated centrosome amplification by targeting at the ROCK1/14-3-3σ complex in human colon cancer HCT116 cells.

We have recently reported that type 2 diabetes promotes centrosome amplification via enhancing the expression, biding, and centrosome translocation of rho‐associated coiled‐coil containing protein kinase 1 (ROCK1)/14‐3‐3σ complex in HCT116 cells. In the functional proteomic study, we further investigated the molecular pathways underlying the centrosome amplification using HCT116 cells. We found that treatment of HCT116 cells with high glucose, insulin, and palmitic acid triggered the centrosome amplification and increased the expressions of proliferating cell nuclear antigen (PCNA), nucleophosmin (NPM), and 14‐3‐3σ. Individual knockdown of PCNA, NPM, or 14‐3‐3σ inhibited the centrosome amplification. Knockdown of PCNA inhibited the treatment‐increased expression of ROCK1, whereas knockdown of ROCK1 did not affect the PCNA expression. High glucose, insulin, and palmitic acid also increased the expressions of c‐Jun N‐terminal kinase‐1 (JNK1) and signal transducer and activator of transcription 3 (Stat3), individual knockdown of which upregulated the treatment‐increased expression of 14‐3‐3σ and promoted the centrosome amplification. In contrast, overexpression of JNK1 inhibited the centrosome amplification. Knockdown of Stat3 enhanced the centrosome translocation of 14‐3‐3σ. Moreover, we showed that knockdown of JNK1 inhibited the treatment‐increased expression of Stat3. Knockdown of PCNA, JNK1, or Stat3 did not have an effect on NPM and vice versa. In conclusion, our results suggest that PCNA and JNK1‐Stat3 pathways respectively promotes and feedback inhibits the centrosome amplification by targeting at the ROCK1/14‐3‐3σ complex, and NPM serves as an independent signal for the centrosome amplification.

ISGF3 with reduced phosphorylation is associated with constitutive expression of interferon-induced genes in aging cells

During cellular aging, many changes in cellular functions occur. A hallmark of aged cells is secretion of inflammatory mediators, which collectively is referred to as the senescence-associated secretory phenotype (SASP). However, the mechanisms underlying such changes are unclear. Canonically, the expression of interferon (IFN)-stimulated genes (ISGs) is induced by IFNs through the formation of the tripartite transcriptional factor ISGF3, which is composed of IRF9 and the phosphorylated forms of STAT1 and STAT2. However, in this study, the constitutive expression of ISGs in human-derived senescent fibroblasts and in fibroblasts from a patient with Werner syndrome, which leads to premature aging, was mediated mainly by the unphosphorylated forms of STATs in the absence of INF production. Under homeostatic conditions, STAT1, STAT2, and IRF9 were localized to the nucleus of aged cells. Although knockdown of JAK1, a key kinase of STAT1 and STAT2, did not affect ISG expression or IFN-stimulated response element (ISRE)-mediated promoter activities in these senescent cells, knockdown of STAT1 or STAT2 decreased ISG expression and ISRE activities. These results suggest that the ISGF3 complex without clear phosphorylation is required for IFN-independent constitutive ISG transcription in senescent cells.

Antisense STAT3 inhibitor decreases viability of myelodysplastic and leukemic stem cells.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.

Long noncoding RNA FAL1 promotes proliferation and inhibits apoptosis of human colon cancer cells.

Aberrant expression of long non-coding RNAs (lncRNAs) has been associated with a variety of malignancies including colon cancer. In this study, we aimed to characterize the biological mechanisms of focally amplified lncRNA on chromosome 1 (FAL1) in colon cancers. Here, our results indicate that FAL1 expression was remarkably up-regulated in colon tumor tissues as compared to corresponding tumor-adjacent normal tissues. Importantly, the cumulative survival rate of patients with high levels of FAL1 in tumor tissues was considerably lower than those with low FAL1 levels in tumor tissues. Cox regression analysis showed that lncRNA FAL1 could act as an independent prognostic factor in CRC. Knockdown of FAL1 in HT29 cells attenuated cell proliferation and stimulated cell apoptosis. In contrast, overmetastasis-related molecules Bcl-2, TGF-β1, p65, and PCNA at the mRNA and protein levels. Mechanistically, FAL1 was found to interact with STAT3 at 200 to 400 bp and promote phosphorylation of STAT3. In addition, we found that knockdown of STAT3 in HT29 cells abolished the effects of FAL1 on cell proliferation as well as the expression of TGF-β1 and Bcl-2. Based on these findings, we concluded that FAL1 might be a potential oncogene for the progression of colon cancer. © 2018 IUBMB Life, 70(11):1093-1100, 2018.

Y-box binding protein-1 and STAT3 independently regulate ATP-binding cassette transporters in the chemoresistance of gastric cancer cells.

Y-box binding protein-1 (YB-1) facilitates cancer chemoresistance through the upregulation of ATP-binding cassette(ABC) transporters associated with multidrug resistance, which is one of the primary obstacles in cancer treatment. Since aberrant Janus kinase (JAK)/signal transducer and activator of transcription(STAT) signaling is also implicated in chemoresistance in numerous human malignancies, the interaction between YB-1 and JAK/STAT signaling was explored underlying the chemoresistance of NUGC3 gastric cancer cells. It was demonstrated that YB-1 translocated into the nuclei of NUGC3 cells exposed to doxorubicin hydrochloride, suggesting its important role in chemoresistance. Consistently, knockdown of YB-1 significantly decreased the chemoresistance of cells to doxorubicin hydrochloride and epirubicin hydrochloride, as evidenced by a decrease in cell viability. Notably, JAK inhibitor AG490 treatment further decreased the cell viability caused by YB-1 inhibition and doxorubicin hydrochloride. It was also observed that YB-1 transcriptionally regulated the ABCC3 transporter, whereas STAT3 modulated ABCC2 transporter levels. These findings suggest that YB-1 and STAT3 act together to facilitate chemoresistance via modulating the expression of different ABC transporters in NUGC3 cells. Notably, siYB-1 did not exhibit any significant effect on STAT3 expression. Similarly, siSTAT3 failed to alter YB-1 expression, suggesting that the two may not regulate each other in a mutual manner. However, double knockdown of YB-1 and STAT3 led to a synergistic inhibition of cell invasion in NUGC3 cells. Nonetheless, the combined treatment of YB-1 antagonists with STAT3 inhibitors may serve as an effective therapy in gastric cancer.

Abstract B20: Premalignant lung lesions demonstrate enhanced PD-L1 upregulation in response to interferon-gamma exposure

Abstract Background: Interferon-gamma (IFN-g) is known to play a pivotal role in PD-L1 expression and immune evasion in cancer cells, but there is little known regarding its interactions with premalignant lung lesions. Methods: Immortalized human bronchial epithelial cells (HBEC-vector control), KRAS-mutated (KRASv12) HBEC cells (HBEC-KRAS), p53 knockdown HBEC cells (HBEC-p53), and p53 knockdown/KRAS mutated cells (HBEC-p53/KRAS) were used to assess mRNA expression as well as surface and total protein expression levels of PD-L1 by RT-PCR, flow cytometry, and Western blot before and after treatment with IFN-g. For STAT-1 knockdown, cells were transiently transfected using Lipofectamine RNAiMAX (Thermo Scientific). After 48 hours of transfection, cells were incubated with IFN-g (50 ng/mL) or PBS with 0.1% BSA for 48 hours, then harvested and analyzed by RT-PCR, flow cytometry, and Western blot. An FFPE tissue block from a patient with known premalignant lesions and lung adenocarcinoma was obtained from the UCLA Lung Cancer Tissue Repository and sectioned to create slides for H&amp;E staining and to identify areas of atypical adenomatous hyperplasia. Serial sectioning was performed to create slides for multiplex staining, targeting anti-PD-L1, anti-p-STAT1-Ser747, and anti-CD8. Quantitative image analysis was performed with Visiopharm. Results: Using RT-PCR, flow cytometry, and Western blot, exposure to IFN-g led to upregulation of PD-L1 in HBECs with various gene mutations (HBEC-vector compared to HBEC-Kras, HBEC-p53, HBEC-KRAS/p53, HBEC-EGFR-L858R) (p&amp;lt;0.05). These experiments demonstrated that PD-L1 is elevated at the mRNA and protein levels with IFN-g exposure, with the greatest upregulation in HBECs with KRAS mutation (p&amp;lt;0.013). To investigate potential mechanisms of PD-L1 upregulation in premalignant cell lines after IFN-g exposure, we evaluated levels of STAT1, pSTAT1, ERK, pERK, AKT, and pAKT in the paired HBEC3 vector and KRAS cell lines. On Western blot, pSTAT1 was the only signaling protein that was upregulated after IFN-g exposure. We then performed STAT-1 knockdown of HBEC3-vector and HBEC3-KRAS cell lines. With IFN-g exposure, there was significantly diminished PD-L1 upregulation in the HBEC3-KRAS with STAT-1 knockdown as compared to the HBEC3-KRAS control (p&amp;lt;0.002). In our human staining, there was increased CD8 T cells (11% versus 2.5%) and pSTAT1 (80% versus 32%) in the vicinity of premalignant lesions as compared to normal tissues. In addition, although we identified sporadic premalignant epithelial cells with membranous PD-L1 staining, no PD-L1 staining was identified in any normal epithelial cells. Conclusions: Premalignant lung lesions may contribute to immune evasion by responding to IFN-gamma exposure with enhanced PD-L1 upregulation via pSTAT1 signaling. Further studies are required to establish the role of IFN-gamma in the immune microenvironment of premalignant lung lesions. Citation Format: Jane Yanagawa, Eileen Fung, Mi-Heon Lee, W. Dean Wallace, Michael C. Fishbein, Manash Paul, Kostyantyn Krysan, John D. Minna, Rong Guo, David Elashoff, Jay M. Lee, Steven M. Dubinett. Premalignant lung lesions demonstrate enhanced PD-L1 upregulation in response to interferon-gamma exposure [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B20.

Epidermal growth factor induces STAT1 expression to exacerbate the IFNr‐mediated PD‐L1 axis in epidermal growth factor receptor‐positive cancers

The epidermal growth factor (EGF) receptor (EGFR) overexpressed in many cancers, including lung and head and neck cancers, and is involved in cancer cell progression and survival. PD-L1, increases in tumor cells to evade and inhibit CD8+ T cells, is a clinical immunotherapeutic target. This study investigated the molecular mechanism of EGF on regulating PD-L1 in EGFR-positive cancers and determined potential agents to reduce PD-L1 expression. RNA sequencing (RNAseq) and bioinformatics analysis were performed to determine potential driver genes that regulate PD-L1 in tumor cells-derived tumorspheres which mimicking cancer stem cells. Then, the specific inhibitors targeting EGFR were applied to reduce the expression of PD-L1 in vitro and in vivo. We validated that EGF could induce PD-L1 expression in the selected EGFR-positive cancers. RNAseq results revealed that STAT1 increased as a driver gene in KOSC-3-derived tumorspheres; these data were analyzed using PANTHER followed by NetworkAnalyst. The blockade of EGFR by afatinib resulted in decreased STAT1 and IRF-1 levels, both are transcriptional factors of PD-L1, and disabled the IFNr-STAT1-mediated PD-L1 axis in vitro and in vivo. Moreover, STAT1 knockdown significantly reduced EGF-mediated PD-L1 expression, and ruxolitinib, a JAK1/JAK2 inhibitor, significantly inhibited STAT1 phosphorylation to reduce the IFNr-mediated PD-L1 axis. These results indicate that EGF exacerbates PD-L1 by increasing the protein levels of STAT1 to enforce the IFNr-JAK1/2-mediated signaling axis in selected EGFR-positive cancers. The inhibition of EGFR by afatinib significantly reduced PD-L1 and may be a potential strategy for enhancing immunotherapeutic efficacy.

STAT3 promotes chronic lymphocytic leukemia progression through upregulating SMYD3 expression.

IntroductionThis study was designed to investigate the roles of STAT3 and SMYD3 in chronic lymphocytic leukemia and the regulatory relationship between STAT3 and SMYD3 in chronic lymphocytic leukemia.Material and methodsThe expression of STAT3 and SMYD3 was determined by RT-qPCR and western blot in chronic lymphocytic leukemia samples and cells (MEC1, CLL). Small interfering RNA was used to knock down the mRNA level of STAT3 and the pcDNA3.1-SMYD3 plasmid was used to construct a SMYD3 overexpression model. An MTT assay was performed to evaluate cell proliferation. A transwell assay was used to detect cell invasion ability. Afterwards, a luciferase reporter assay and chromatin immunoprecipitation experiment (ChIP assay) were applied to confirm the correlation between STAT3 and SMYD3.ResultsSTAT3 was highly expressed in chronic lymphocytic leukemia mononuclear cells and cancerous cell lines. STAT3 knockdown dramatically inhibited the mRNA and protein expression of SMYD3 in MEC1 and CLL cell lines. The luciferase reporter assay combined with the ChIP assay revealed that STAT3 bound to the promoter region of STAT3 and contributed to the transcription of SMYD3. Knockdown of STAT3 positively correlated with inhibition of cell proliferation and invasion abilities, while overexpression of SMYD3 negatively correlated with inhibition of cell proliferation and invasion.ConclusionsSTAT3 may promote chronic lymphocytic leukemia progression through elevating SMYD3 expression. Targeting STAT3 and SMYD3 may be a potential therapeutic strategy for chronic lymphocytic leukemia.

Computational Analysis of Transcriptional Regulation Sites at the HTT Gene Locus

Huntington's disease is a late onset neurological disorder caused by a trinucleotide CAG repeat expansion mutation in the HTT gene encoding for the protein huntingtin. Despite considerable ongoing research, the wild-type function of huntingtin is not yet fully understood. To improve knowledge of HTT gene regulation at the transcriptional level and inform future studies aimed at uncovering the HTT gene's normal function. The HTT gene region was functionally characterized through an in silico analysis using publicly available data sets. ChIP-seq data sets and the online STRING database were used to identify putative transcription factor binding sites (TFBSs) and protein-protein interactions within the HTT promoter region. siRNA-mediated knockdown and ChIP-qPCR of STAT1, a TF identified from the in silico analysis, were used to validate the bioinformatics screen. 16 regions containing potential regulatory genomic markers were identified. TFBSs for 59 transcription factors (TFs) were detected in one or more of the 16 candidate regions. Using these TFs, 15 clusters of protein-protein interactions were identified using STRING. siRNA-mediated knockdown of STAT1 resulted in an increase in HTT expression, and ChIP-qPCR detected enrichment of STAT1 binding at one of the predicted regions. These assays confirmed the utility of the bioinformatic analysis. Putative regulatory regions outside of the immediate HTT promoter region have been identified with specific protein-protein interactions. Future work will focus on in vitro and in vivo studies to examine the effect of modulating identified TFBSs and altering the levels of specific TFs of interest in regulating HTT gene expression.