Coactivation Of STAT3 Research Articles

PAR1 and PAR4 exert opposite effects on tumor growth and metastasis of esophageal squamous cell carcinoma via STAT3 and NF-\u03baB signaling pathways

BackgroundEsophageal carcinogenesis is a multifactorial process in which genetic and environmental factors interact to activate intracellular signals, leading to the uncontrolled survival and growth of esophageal squamous cell carcinoma (ESCC) cells. The intracellular pathways of ESCC cells could be regulated by proteinase activated-receptors (PARs), which are comprised of four receptors (i.e., PAR-1, PAR-2, PAR-3, and PAR-4). Therefore, the function and possible mechanism of PAR1 and PAR4 in the progression of ECSS were explored in our study.MethodsFirst, we detected the expression levels of PAR1 and PAR4 in 27 cases of ESCC specimens and cell lines by RT-qPCR, IHC and western blot. Meanwhile, the correlation between PAR1/PAR4 expression levels, clinicopathological characteristics, and disease free survival was analyzed. Then, we constructed PAR1/PAR4 knockdown cell models and investigated the role of PAR1/PAR4 knockdown on the proliferation, apoptosis, changes of calcium flow, and metastasis of ESCC cells via MTT, flow cytometry, transwell and wound healing assays in vitro. Further, an experimental metastasis model in vivo was established to explore the role of stable PAR1/PAR4 knockdown on the growth and metastasis of ESCC cells. Finally, the role of nSMase2 in the activation of NF-κB induced by PAR4 and the role of NF-κB and STAT3 signaling pathways in the PAR1/PAR4-mediated tumor promoting or suppressive functions were measured by immunoprecipitation, western blot and immunofluorescence assays.ResultsFirst, the integrated results demonstrated the expression levels of PAR1 and PAR4 are inversely proportional in ESCC. PAR1 potently enhanced tumor growth and metastasis, while PAR4 had an inhibitory effect. Further, the co-activation of STAT3 and NF-κB was involved in the PAR1 activation-induced tumor promoting effect, while only NF-κB participated in the PAR4 activation-induced tumor inhibitory effect in ESCC. To be specific, FAK/PI3K/AKT/STAT3/NF-κB signaling mediated PAR1 activation-induced tumor promoting effect and nSMase2/MAPK/NF-κB signaling mediated PAR4 activation-induced tumor inhibitory effect.ConclusionsOverall, the study has provided new insights into the potential implication of PAR1 and PAR4 in the pathogenesis of ESCC. Besides, FAK/PI3K/AKT/STAT3/NF-κB and nSMase2/MAPK/NF-κB pathways may be novel targets for regulating tumor growth and metastasis in ESCC patients.

CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

BackgroundSignal transducer and activator of transcription 3 (STAT3) has been shown to upregulate gene transcription during tumorigenesis. However, how STAT3 initiates transcription remains to be exploited. This study is to reveal the role of CREPT (cell cycle-related and elevated-expression protein in tumours, or RPRD1B) in promoting STAT3 transcriptional activity.MethodsBALB/c nude mice, CREPT overexpression or deletion cells were employed for the assay of tumour formation, chromatin immunoprecipitation, assay for transposase-accessible chromatin using sequencing.ResultsWe demonstrate that CREPT, a recently identified oncoprotein, enhances STAT3 transcriptional activity to promote tumorigenesis. CREPT expression is positively correlated with activation of STAT3 signalling in tumours. Deletion of CREPT led to a decrease, but overexpression of CREPT resulted in an increase, in STAT3-initiated tumour cell proliferation, colony formation and tumour growth. Mechanistically, CREPT interacts with phosphorylated STAT3 (p-STAT3) and facilitates p-STAT3 to recruit p300 to occupy at the promoters of STAT3-targeted genes. Therefore, CREPT and STAT3 coordinately facilitate p300-mediated acetylation of histone 3 (H3K18ac and H3K27ac), further augmenting RNA polymerase II recruitment. Accordingly, depletion of p300 abolished CREPT-enhanced STAT3 transcriptional activity.ConclusionsWe propose that CREPT is a co-activator of STAT3 for recruiting p300. Our study provides an alternative strategy for the therapy of cancers related to STAT3.

Abstract 5415: STAT3 and GLI1/tGLI1 oncogenic transcription factors interact to promote the aggressiveness of triple-negative and HER2-enriched breast cancers

Abstract Patients with triple-negative (TN) and HER2-enriched (HER2) breast cancers have high rates of metastasis, which accounts for 90% mortality for these patients, highlighting the need for improved therapeutic targets. The oncogenic transcription factors, STAT3, GLI1, and truncated GLI1 (tGLI1) are associated with poor clinical outcomes for breast cancer patients. However, physical or functional interactions between STAT3 and GLI1/tGLI1 have never been investigated. The purpose of this study is to determine whether the JAK2/STAT3 and GLI1/tGLI1 pathways are concurrently activated in TN and HER2 breast cancers. We examined 47 node-positive breast tumor samples using immunohistochemical staining and found that p-STAT3 (Y705), GLI1, and tGLI1 were co-overexpressed in 64% of TN breast cancers, 68% of HER2 breast cancers, and in 65% of lymph node metastases. Gene Set Enrichment Analysis of 710 breast tumors revealed that the activation signatures for STAT3 and GLI1/tGLI1 are co-enriched in TN and HER2 subtypes, but not in the luminal subtypes. Furthermore, Kaplan-Meier and log-rank analyses revealed that breast tumors with high levels of STAT3 and GLI1/tGLI1 co-activation were associated with shortened metastasis-free survival compared to those with lower levels. To determine whether these two pathways functionally cooperate, we examined whether STAT3 and GLI1/tGLI1 formed complexes by IP-WB and observed that STAT3 interacted with both GLI1 and tGLI1. ChIP assay showed that these complexes bind to both consensus GLI1- and STAT3-binding sites. We further observed that co-overexpression of STAT3 and GLI1/tGLI1 resulted in significant activation of a promoter controlled by GLI1-binding sites. We next analyzed three ChIP-Seq datasets to identify gene promoters that can be occupied by the STAT3-GLI1/tGLI1 complexes, and found 36 potential target genes. Validation of these target genes by RT-qPCR and Kaplan-Meier analysis resulted in three genes that are upregulated by STAT3-GLI1 and/or STAT3-tGLI1, namely, R-Ras2, Cep70, and UPF3A, and correlated with poor survival. These novel transcriptional targets of STAT3 and GLI1/tGLI1 are implicated in breast tumorigenesis, angiogenesis, and metastasis suggesting that co-activation of STAT3 and GLI1/tGLI1 may promote aggressive breast cancers. Lastly, co-overexpression of STAT3 and GLI1/tGLI1 increased the mammosphere-forming ability of breast cancer cells and interestingly, only the STAT3-tGLI1 complex allowed mammosphere formation of immortalized mammary epithelial cells. In conclusion, our study reports, for the first time, that the physical and functional interactions between STAT3 and GLI1/tGLI1 oncogenic transcription factors lead to gene co-activation, stem-like phenotype of breast cancer cells, and unfavorable prognosis for patients with triple-negative and HER2 breast cancers. Citation Format: Sherona R. Sirkisoon, Richard L. Carpenter, Rimkus Tadas, Anderson Ashley, Alexandria Harrison, Allison M. Lange, Guangxu Jin, Kounosuke Watabe, Hui-Wen Lo. STAT3 and GLI1/tGLI1 oncogenic transcription factors interact to promote the aggressiveness of triple-negative and HER2-enriched breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5415.

Abstract 2540: TRIM24 is an oncogenic transcriptional co-activator of STAT3 in glioblastoma

Abstract Aberrant amplification and mutations of epidermal growth factor receptor (EGFR) are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive pathogenesis are not well understood. Here, we determine that non-canonical histone signature acetylated H3 lysine 23 (H3K23ac)-binding protein tripartite motif-containing 24 (TRIM24) is upregulated in clinical GBM specimens and required for EGFR-driven tumorigenesis. In multiple glioma cell lines and patient-derived glioma stem cells (GSCs), EGFR signaling promotes H3K23 acetylation and association with TRIM24. Consequently, TRIM24 functions as a transcriptional co-activator and recruits STAT3, leading to stabilized STAT3-chromatin interactions and subsequent activation of STAT3 downstream signaling, thereby enhancing EGFR-driven tumorigenesis. Our findings uncover a pathway in which TRIM24 functions as a signal relay for oncogenic EGFR signaling and suggest TRIM24 as a potential therapeutic target for GBM that are associated with EGFR activation. Citation Format: Deguan Lv, Yanxin Li, Weiwei Zhang, Angel A. Alvarez, Jianming Tang, Bo Hu, Shi-Yuan Cheng, Haizhong Feng. TRIM24 is an oncogenic transcriptional co-activator of STAT3 in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2540.

The DEAD box protein p68: a novel coactivator of Stat3 in mediating oncogenesis.

DEAD box RNA helicase p68 acts as a transcriptional coactivator of several oncogenic transcription factors apart from being a vital player of RNA metabolism. Signal transducer and activator of transcription 3 (Stat3) is a major oncogenic contributor of diverse cancers, including that of colon. Deciphering the mechanistic insights of coactivation of Stat3 transcriptional activity may aid in improved therapeutic strategies. Here we report for the first time a novel mechanism of alliance between p68 and Stat3 in stimulating transcriptional activity of Stat3. Interestingly, we observed that the expression of p68 and Stat3 bears strong positive correlation and significant colocalization in normal and colon carcinoma patient samples. We demonstrated that p68 directly interacts with Stat3 in HEK293 cells as well as multiple colon cancer cell lines. Additionally, p68 positively modulated both mRNA and protein expression levels of Stat3 target genes; promoter activity of Stat3 target gene Mcl-1 in multiple colon cancer cell lines. Also, p68 occupied the promoters of multiple Stat3 target genes in enhancing Stat3-dependent transcription. Moreover, the strong positive correlation between the abundance of p68 and Stat3 target genes in the same set of colon carcinoma samples further supported our observations. Enhanced expression levels of Stat3 target genes observed in primary tumors and metastatic lung nodules, generated in mice colorectal allograft model using syngeneic cells stably expressing p68, further reinforced our in vitro findings. Hence, this study unravels novel modes of p68-mediated oncogenesis through coactivation of Stat3 and enhancing Stat3 signaling.

Mechanism of androgen receptor corepression by CKβBP2/CRIF1, a multifunctional transcription factor coregulator expressed in prostate cancer

The transcription factor coregulator Casein kinase IIβ-binding protein 2 or CR6-interacting factor 1 (CKβBP2/CRIF1) binds the androgen receptor (AR) in prostate cancer cells and in response to dihydrotestosterone localizes with AR on the prostate-specific antigen gene enhancer, but does not bind DNA suggesting CKβBP2/CRIF1 localization in chromatin is determined by AR. In this study we show also that CKβBP2/CRIF1 inhibits wild-type AR and AR N-terminal transcriptional activity, binds to the AR C-terminal region, inhibits interaction of the AR N- and C-terminal domains (N/C interaction) and competes with p160 coactivator binding to the AR C-terminal domain, suggesting CKβBP2/CRIF1 interferes with AR activation functions 1 and 2. CKβBP2/CRIF1 is expressed mainly in stromal cells of benign prostatic hyperplasia and in stroma and epithelium of prostate cancer. CKβBP2/CRIF1 protein is increased in epithelium of androgen-dependent prostate cancer compared to benign prostatic hyperplasia and decreased slightly in castration recurrent epithelium compared to androgen-dependent prostate cancer. The multifunctional CKβBP2/CRIF1 is a STAT3 interacting protein and reported to be a coactivator of STAT3. CKβBP2/CRIF1 is expressed with STAT3 in prostate cancer where STAT3 may help to offset the AR repressor effect of CKβBP2/CRIF1 and allow AR regulation of prostate cancer growth.

Progestin drives breast cancer growth by inducing p21CIP1 expression through the assembly of a transcriptional complex among Stat3, progesterone receptor and ErbB-2

Cell cycle regulator p21CIP1 has controversial biological effects in breast cancer since in spite of its role as cell cycle inhibitor and promoter of cellular senescence, it also induces cell proliferation and chemoteraphy resistance. We here explored the molecular mechanisms involved in progestin regulation of p21CIP1 expression. We also investigated the biological effects of p21CIP1 in breast cancer cells. We found that the synthetic progestin medroxyprogesterone acetate (MPA) upregulates p21CIP1 protein expression via c-Src, signal transducer and activator of transcription 3 (Stat3) and ErbB-2 phosphorylation. Notably, we also found that ErbB-2 nuclear function plays a key role in MPA-induction of p21CIP1 expression. Interestingly, we determined that progestin drives p21CIP1 transcriptional activation via a novel nonclassical transcriptional mechanism in which progesterone receptor is recruited along with Stat3 and ErbB-2 to a Stat3 binding site at p21CIP1 promoter. Our findings revealed that ErbB-2 functions as a coactivator of Stat3 in progestin induction of p21CIP1 transcriptional activation. Furthermore, we demonstrated that blockage of p21CIP1 expression strongly inhibited in vitro and in vivo progestin-induced breast cancer cell proliferation. These results further support the hypothesis that according to cell context and type of stimulus, p21CIP1 is capable of inducing cell cycle progression. Moreover, we provided evidence that Stat3 and nuclear ErbB-2 are key players in progestin-induced p21CIP1 regulation.

Suppression of FoxO1 Activity by Long-Chain Fatty Acyl Analogs

OBJECTIVEOveractivity of the Forkhead transcription factor FoxO1 promotes diabetic hyperglycemia, dyslipidemia, and acute-phase response, whereas suppression of FoxO1 activity by insulin may alleviate diabetes. The reported efficacy of long-chain fatty acyl (LCFA) analogs of the MEDICA series in activating AMP-activated protein kinase (AMPK) and in treating animal models of diabesity may indicate suppression of FoxO1 activity.RESEARCH DESIGN AND METHODSThe insulin-sensitizing and anti-inflammatory efficacy of a MEDICA analog has been verified in guinea pig and in human C-reactive protein (hCRP) transgenic mice, respectively. Suppression of FoxO1 transcriptional activity has been verified in the context of FoxO1- and STAT3-responsive genes and compared with suppression of FoxO1 activity by insulin and metformin.RESULTSTreatment with MEDICA analog resulted in total body sensitization to insulin, suppression of lipopolysaccharide-induced hCRP and interleukin-6–induced acute phase reactants and robust decrease in FoxO1 transcriptional activity and in coactivation of STAT3. Suppression of FoxO1 activity was accounted for by its nuclear export by MEDICA-activated AMPK, complemented by inhibition of nuclear FoxO1 transcriptional activity by MEDICA-induced C/EBPβ isoforms. Similarly, insulin treatment resulted in nuclear exclusion of FoxO1 and further suppression of its nuclear activity by insulin-induced C/EBPβ isoforms. In contrast, FoxO1 suppression by metformin was essentially accounted for by its nuclear export by metformin-activated AMPK.CONCLUSIONSSuppression of FoxO1 activity by MEDICA analogs may partly account for their antidiabetic anti-inflammatory efficacy. FoxO1 suppression by LCFA analogs may provide a molecular rational for the beneficial efficacy of carbohydrate-restricted ketogenic diets in treating diabetes.

Progesterone Receptor Induces ErbB-2 Nuclear Translocation To Promote Breast Cancer Growth via a Novel Transcriptional Effect: ErbB-2 Function as a Coactivator of Stat3

Progesterone receptor (PR) and ErbB-2 bidirectional cross talk participates in breast cancer development. Here, we identified a new mechanism of the PR and ErbB-2 interaction involving the PR induction of ErbB-2 nuclear translocation and the assembly of a transcriptional complex in which ErbB-2 acts as a coactivator of Stat3. We also highlighted that the function of ErbB-2 as a Stat3 coactivator drives progestin-induced cyclin D1 promoter activation. Notably, PR is also recruited together with Stat3 and ErbB-2 to the cyclin D1 promoter, unraveling a new and unexpected nonclassical PR genomic mechanism. The assembly of the nuclear Stat3/ErbB-2 transcriptional complex plays a key role in the proliferation of breast tumors with functional PR and ErbB-2. Our findings reveal a novel therapeutic intervention for PR- and ErbB-2-positive breast tumors via the specific blockage of ErbB-2 nuclear translocation.

SS2-8 Gastric tumour progression in gp130Y757F mice is dependent on co-activation of Stat3 and mTOR via Interleukin-11 signaling

SS2-8 Gastric tumour progression in gp130Y757F mice is dependent on co-activation of Stat3 and mTOR via Interleukin-11 signaling

Abstract 1718: ErbB-2 acts as coactivator of Stat3 in heregulin-induced breast cancer growth

Abstract We have previously demonstrated that heregulin (HRG), a ligand for ErbB receptors, activates signal transducer and activator of transcription 3 (Stat3) in primary cultures of ErbB-2 overexpressing C4HD cells, from a murine progestin-dependent mammary tumor. ErbB-2 activity is an absolute requirement in the mechanisms of HRG stimulation of Stat3 activity. Recent findings have demonstrated ErbB-2 nuclear migration and its function as a transcription factor. In this work, we studied whether HRG induces ErbB-2 nuclear migration and its interaction with Stat3 in breast cancer cells. We demonstrated that HRG induces ErbB-2 nuclear migration and colocalization with Stat3 in C4HD and human T47D breast cancer cells. Cyclin D1 is a cancer-related gene that contains Stat3 binding sites (GAS sites) but lacks ErbB-2 response elements (HAS sites). By chromatin immunoprecipitation assays (ChIP) and sequential ChIP, we demonstrated that HRG induces in vivo binding of Stat3 and ErbB-2 to the GAS sites of the cyclin D1 promoter. This finding prompted us to evaluate the ability of HRG to regulate cyclin D1 expression. By inhibiting ErbB-2 and Stat3 activation or silencing their expression, we demonstrated that ErbB-2 and Stat3 participate in the mechanism of HRG-induced cyclin D1 protein expression. Next, we explored whether HRG induces cyclin D1 promoter directly via Stat3 binding to its response elements. C4HD and T47D cells transiently transfected with a cyclin D1 promoter luciferase construct showed an enhanced transcriptional activity with HRG treatment. Overexpression of increasing amounts of ErbB-2 wt resulted in a dose-dependent ErbB-2 capacity to enhance Stat3 transcriptional activity induced by HRG. On the other hand, transfection with increasing amounts of an ErbB-2 mutant that is defective in nuclear entry but retains its cell-surface location and functions (ErbB-2ΔNLS) resulted in abrogation of HRG-induced Stat3 activation of cyclin D1 promoter. Finally, we addressed the effect of targeting ErbB-2 in in vivo HRG-dependent growth of C4HD breast tumors. Transfection of C4HD cells with the ErbB-2ΔNLS expression vector significantly inhibited these cells’ ability to form tumors in syngeneic mice. Taken together, these results suggest a new role of ErbB-2 as a coactivator in the mechanism of HRG-induced transcriptional activation of Stat3. We also found nuclear ErbB-2 to be a requisite for HRG stimulation of in vitro and in vivo breast cancer growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1718.

Abstract 4614: Progesterone receptor and ErbB-2 crosstalk in breast cancer

Abstract Progesterone receptor (PR) and ErbB-2, a member of the ErbBs family of receptor tyrosine kinases, play key roles in breast cancer development. Here, we identified a new mechanism of PR and ErbB-2 interaction in breast cancer cells. Our findings demonstrated that progestin stimulates the rapid activation of ErbB-2, its nuclear translocation, and a striking nuclear colocalization of ErbB-2 with signal transducer and activator of transcription 3 (Stat3) in human T47D breast cancer cells and C4HD cells, from a murine progestin-dependent mammary tumor. By using quantitative chromatin immunoprecipitation (ChIP) and sequential ChIP, we demonstrated that progestin induces the recruitment of Stat3 and ErbB-2 to Stat3 response elements (GAS sites) in the cyclin D1 promoter, which lacks ErbB-2 response elements (HAS sites). Through a series of experimental approaches including transient transfection with cyclin D1 promoter luciferase constructs and RNA interfering-reconstitution strategies employing functional ErbB-2 and Stat3 mutants, we revealed ErbB-2 role as a transcriptional coactivator of Stat3 in progestin-induced cyclin D1 promoter activation. Inhibition of ErbB-2 nuclear migration by transfection of breast cancer cells with the ErbB-2 nuclear localization domain mutant ErbB-2ΔNLS (Mol Cell Biol, 24: 11005-11018, 2005), which we here found that functions as a dominant negative inhibitor of endogenous ErbB-2 nuclear translocation, resulted in complete abrogation of progestin-induced in vitro and in vivo breast cancer growth. Our findings reveal a novel therapeutic intervention in PR- and ErbB-2- positive breast tumors via the specific blockage of ErbB-2 nuclear translocation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4614.

Expression of Stat3 and Notch1 is associated with cisplatin resistance in head and neck squamous cell carcinoma

Cisplatin is the most important chemotherapeutic agent involved in treatment of head and neck squamous cell carcinoma (HNSCC), but cisplatin resistance in HNSCC is still a serious problem in clinic. The reasons why patients fail chemotherapy are unclear. We examined 25 HNSCC patients who were all tested for cisplatin sensitivity by CD-DST (collagen gel droplet embedded culture-drug sensitivity) method and expression of Stat3 and Notch1. We found that high expression levels of Stat3 and Notch1 were closely associated with cisplatin resistance respectively (P=0.014, P=0.000). In addition, cisplatin resistance of HNSCC was decreased after inhibition of Stat3 or Notch signaling in vitro. Our results provide first evidence that both high Stat3 and Notch1 expression are associated with cisplatin resistance in HNSCC patients, supporting the hypothesis that co-activation of Stat3 and Notch1 by their crosstalk induces the reprogrammed survival pathways in HNSCC responding to chemotherapy.

Akt Modulates STAT3-mediated Gene Expression through a FKHR (FOXO1a)-dependent Mechanism

The phosphatidylinositol 3-kinase/Akt pathway plays an important role in the signaling of insulin and other growth factors, which reportedly attenuate the interleukin-6 (IL-6)-mediated stimulation of acute phase plasma protein genes. We investigated the effect of the protein kinase Akt on IL-6-mediated transcriptional activation. The transient expression of constitutively active Akt inhibited the IL-6-dependent activity of the alpha(2)-macroglobulin promoter in HepG2 cells, whereas expression of an inactive mutant of phosphatidylinositol-dependent kinase 1 had the opposite effect. Since Akt is known to regulate gene expression through inactivation of the transcription factor FKHR (forkhead in rhabdomyosarcoma), we examined the effect of FKHR on STAT3-mediated transcriptional regulation. Indeed, the overexpression of FKHR specifically enhanced the activity of STAT3-dependent promoters but not that of a STAT5-responsive promoter. The effect of FKHR required the presence of functional STAT3 and was abrogated by the expression of dominant negative STAT3 mutants. Furthermore, FKHR and STAT3 were shown to coimmunoprecipitate and to colocalize in the nuclear regions of IL-6-treated HepG2 cells. Our results indicate that FKHR can modulate the IL-6-induced transcriptional activity by acting as a coactivator of STAT3.