Signal Transducer And Activator Of Transcription 3 Promoter Research Articles

Zinc finger protein 263 promotes colorectal cancer cell progression by activating STAT3 and enhancing chemoradiotherapy resistance

Zinc finger protein 263 (ZNF263) is frequently upregulated in various tumor types; however, its function and regulatory mechanism in colorectal cancer (CRC) have not yet been elucidated. In this study, the expression of ZNF263 was systematically examined using data from The Cancer Genome Atlas database and samples from patients with CRC. The results indicated that high expression of ZNF263 in CRC tissues is significantly associated with tumor grade, lymph node metastasis and disant metastasis. Additionally, overexpression of ZNF263 significantly promoted the proliferation, invasion, migration, and epithelial-mesenchymal transition of CRC cells, while also increasing signal transducer and activator of transcription 3 (STAT3) expression and mRNA stability. Conversely, knockdown of ZNF263 inhibited the malignant behavior of CRC cells and decreased STAT3 expression and mRNA stability. Further mechanism studies using chromatin immunoprecipitation (CHIP) and luciferase assays verified that ZNF263 directly binds to the STAT3 promoter. Rescue experiments demonstrated that the knockdown or overexpression of STAT3 could significantly reverse the effects of ZNF263 on CRC cells. Additionally, our study found that overexpression of ZNF263 enhanced the resistance of CRC cells to the chemoradiotherapy. In summary, this study not only elucidated the significant role of ZNF263 in CRC but also proposed novel approaches and methodologies for the diagnosis and treatment of this malignancy.

HMGA2 facilitates colorectal cancer progression via STAT3-mediated tumor-associated macrophage recruitment.

Rationale: Tumor-associated macrophages (TAMs), generally displaying the pro-tumor M2-like phenotype, strongly influence the progression of colorectal cancer (CRC) via their immunosuppressive activities. The high-mobility gene group A2 (HMGA2), an oncoprotein, is aberrantly overexpressed in CRC cells. However, the mechanisms by which tumor-derived HMGA2 modulates tumor microenvironment in CRC remain poorly understood.Methods: In vivo subcutaneous tumor xenograft model, azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced tumor mouse model and in vitro co-culture assays were used to investigate the Hmga2 role in TAM recruitment and polarization. Luciferase and chromatin immunoprecipitation (ChIP) assays were applied to examine the mechanism of HMGA2-mediated transcriptional regulation of signal transducer and activator of transcription 3 (STAT3). The CD68 correlation with patient outcome was analyzed in 167 human CRC tissues.Results: We found that HMGA2 in cancer cells promoted macrophage recruitment and M2 polarization in vitro and in vivo. HMGA2 directly bound to the STAT3 promoter to activate its transcription and subsequently induced CCL2 secretion, thus promoting macrophage recruitment. Our results from human CRC specimens also revealed a strong positive association between HMGA2 expression in tumor cells and CD68 expression in the stroma. We further showed that patients with an elevated CD68 expression had an unfavorable overall survival in all of the patients or in the subgroup with negative distant metastasis.Conclusion: Our work uncovers new insight into the link between the HMGA2/STAT3/CCL2 axis and macrophage recruitment in CRC. These findings provide a novel therapeutic option for targeting the HMGA2/STAT3/CCL2 axis in CRC.

Fat mass and obesity-associated protein (FTO) mediates signal transducer and activator of transcription 3 (STAT3)-drived resistance of breast cancer to doxorubicin

ABSTRACT Excessive activation of signal transducer and activator of transcription 3 (STAT3) is implicated in breast cancer (BC) chemoresistance, but its underlying mechanism is not fully understood. There are STAT3 binding sites in fat mass and obesity-associated protein (FTO) promoter region, thus STAT3 may regulate the transcription of FTO. This study aimed to investigate the correlation between FTO and STAT3 in BC chemoresistance. Herein, FTO and STAT3 were highly expressed in doxorubicin-resistant BC (BC-DoxR) cells. CHIP assay verified the binding between STAT3 and FTO promoter in BC-DoxR cells. Dual luciferase reporter assay showed that FTO promoter activity was inhibited by S3I-201 (STAT3 inhibitor) but enhanced by epidermal growth factor (EGF, STAT3 activator) in BC-DoxR and BC cells. FTO mRNA and protein expression were suppressed by S3I-201 in BC-DoxR cells and EGF-stimulated BC cells. Notably, FTO regulated total N6-methyladenosine (m6A) levels in BC-DoxR and BC cells but could not affect STAT3 mRNA expression, indicating that FTO was not involved in the m6A modification of STAT3. However, FTO could activate STAT3 signaling in BC-DoxR and BC cells. Besides, FTO knockdown inhibited the doxorubicin resistance of BC-DoxR cells, while FTO overexpression enhanced the doxorubicin resistance and weakened the doxorubicin sensitivity of BC cells. Moreover, decreased doxorubicin resistance by STAT3 knockdown was abolished by FTO overexpression and decreased doxorubicin sensitivity by STAT3 overexpression was reversed by FTO knockdown, indicating that FTO was implicated in STAT3-mediated doxorubicin resistance and impairment of doxorubicin sensitivity of BC cells. Altogether, our findings provide a mechanism underlying BC doxorubicin resistance.

Antitumor activity of a novel oral signal transducer and activator of transcription 3 inhibitor YHO-1701.

The signal transducer and activator of transcription 3 (STAT3) signaling pathway is a key mediator of cancer cell proliferation, survival and invasion. Aberrant STAT3 has been demonstrated in various malignant cancers. YHO‐1701 is a novel quinolinecarboxamide derivative generated from STX‐0119. Here, we examined the effect of YHO‐1701 on STAT3 and evaluated antitumor activity of YHO‐1701 as a single agent and in combination. YHO‐1701 inhibited STAT3‐SH2 binding to phospho‐Tyr peptide selectively and more potently than STX‐0119 in biochemical assays. Molecular docking studies with STAT3 suggested more stable interaction of YHO‐1701 with the SH2 domain. YHO‐1701 exhibited approximately 10‐fold stronger activity than STX‐0119 in abrogating the STAT3 signaling pathway of human oral cancer cell line SAS. YHO‐1701 also blocked multi‐step events by inhibiting STAT3 dimerization and suppressed STAT3 promoter activity. As expected, YHO‐1701 exerted strong antiproliferative activity against human cancer cell lines addicted to STAT3 signaling. Orally administered YHO‐1701 showed statistically significant antitumor effects with long exposure to high levels of YHO‐1701 at tumor sites in SAS xenograft models. Moreover, combination regimen with sorafenib led to significantly stronger antitumor activity. In addition, the suppression level of survivin (a downstream target) was superior for the combination as compared with monotherapy groups within tumor tissues. Thus, YHO‐1701 had a favorable specificity for STAT3 and pharmacokinetics after oral treatment; it also contributed to the enhanced antitumor activity of sorafenib. The evidence presented here provides justification using for this approach in future clinical settings.

Nicotine Upregulates the Level of Mcl-1 through STAT3 in H1299 Cells.

Background: Nicotine contributes to development of human lung cancer and chemoresistance through activation of myeloid cell leukemia-1 (Mcl-1). Signal transducer and activator of transcription 3 (STAT3) generally participates in development and progression of human cancers. Therefore, we examined the STAT3 cascade in nicotine regulation of Mcl-1 transcription in human lung cancer cells.Methods: The effects of nicotine on the expression of STAT3 and Mcl-1 were determined using western blot. The sub-cellular localization was tested using immunofluorescence. The activity of STAT3 promoter was checked using dual luciferase reporter assay.Results: STAT3 was constitutively activated (i.e., tyrosine-phosphorylated, serine-phosphorylated and nuclear translocation), meanwhile the expression and transcriptional activity of Mcl-1 were up-regulated in lung cancer cells following treatment with nicotine. Transfection with siRNA targeting STAT3 or treatment with STAT3 inhibitor JSI-124 diminished Mcl-1 protein levels. Deleted mutagenesis of a putative STAT3 consensus binding sequence decreased Mcl-1 promoter activity and eliminated the increase of Mcl-1 promoter activity induced by nicotine. Abnormally, JAK (Jannus kinase) inhibitor AG490 can't induce the downregulation of Mcl-1 or inhibit the tyrosine-phosphorylation of STAT3. In addition, deactivated mutagenesis of STAT3 the tyrosine 705 site had no effect on the aggregation of STAT3 into nucleus induced by nicotine.Conclusions: We have demonstrated that nicotine induces up-regulation of Mcl-1 through STAT3, which process may be independent on JAKs and not only dependent on the phosphorylation of Y705. Downregulation of Mcl-1 transcription by inhibiting STAT3 cascade may be a potential strategy for the treatment of this cancer.

C/EBPβ Promotes STAT3 Expression and Affects Cell Apoptosis and Proliferation in Porcine Ovarian Granulosa Cells.

Previous studies suggest that signal transducer and activator of transcription 3 (STAT3) and CCAAT/enhancer binding protein beta (C/EBPβ) play an essential role in ovarian granulosa cells (GCs) for mammalian follicular development. Several C/EBPβ putative binding sites were previously predicted on the STAT3 promoter in mammals. However, the molecular regulation of C/EBPβ on STAT3 and their effects on cell proliferation and apoptosis remain virtually unexplored in GCs. Using porcine GCs as a model, the 5′-deletion, luciferase report assay, mutation, chromatin immunoprecipitation, Annexin-V/PI staining and EdU assays were applied to investigate the molecular mechanism for C/EBPβ regulating the expression of STAT3 and their effects on the cell proliferation and apoptosis ability. We found that over and interfering with the expression of C/EBPβ significantly increased and decreased the messenger RNA (mRNA) and protein levels of STAT3, respectively. The dual luciferase reporter assay showed that C/EBPβ directly bound at −1397/−1387 of STAT3 to positively regulate the mRNA and protein expressions of STAT3. Both C/EBPβ and STAT3 were observed to inhibit cell apoptosis and promote cell proliferation. Furthermore, C/EBPβ might enhance the antiapoptotic and pro-proliferative effects of STAT3. These results would be of great insight in further exploring the molecular mechanism of C/EBPβ and STAT3 on the function of GCs and the development of ovarian follicles in mammals.

ZNF341 controls STAT3 expression and thereby immunocompetence.

Signal transducer and activator of transcription 3 (STAT3) is a central regulator of immune homeostasis. STAT3 levels are strictly controlled, and STAT3 impairment contributes to several diseases including the monogenic autosomal-dominant hyper-immunoglobulin E (IgE) syndrome (AD-HIES). We investigated patients of four consanguineous families with an autosomal-recessive disorder resembling the phenotype of AD-HIES, with symptoms of immunodeficiency, recurrent infections, skeletal abnormalities, and elevated IgE. Patients presented with reduced STAT3 expression and diminished T helper 17 cell numbers, in absence of STAT3 mutations. We identified two distinct homozygous nonsense mutations in ZNF341, which encodes a zinc finger transcription factor. Wild-type ZNF341 bound to and activated the STAT3 promoter, whereas the mutant variants showed impaired transcriptional activation, partly due to nuclear translocation failure. In summary, nonsense mutations in ZNF341 account for the STAT3-like phenotype in four autosomal-recessive kindreds. Thus, ZNF341 is a previously unrecognized regulator of immune homeostasis.

Genetic Variants in STAT3 Promoter Regions and Their Application in Molecular Breeding for Body Size Traits in Qinchuan Cattle.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in leptin-mediated regulation of energy metabolism. This study investigated genetic variation in STAT3 promoter regions and verified their contribution to bovine body size traits. We first estimated the degree of conservation in STAT3, followed by measurements of its mRNA expression during fetal and adult stages of Qinchuan cattle. We then sequenced the STAT3 promoter region to determine genetic variants and evaluate their association with body size traits. From fetus to adult, STAT3 expression increased significantly in muscle, fat, heart, liver, and spleen tissues (p < 0.01), but decreased in the intestine, lung, and rumen (p < 0.01). We identified and named five single nucleotide polymorphisms (SNPs): SNP1-304A>C, SNP2-285G>A, SNP3-209A>C, SNP4-203A>G, and SNP5-188T>C. These five mutations fell significantly outside the Hardy–Weinberg equilibrium (HWE) (Chi-squared test, p < 0.05) and significantly associated with body size traits (p < 0.05). Individuals with haplotype H3H3 (CC-GG-CC-GG-CC) were larger in body size than other haplotypes. Therefore, variations in the STAT3 gene promoter regions, most notably haplotype H3H3, may benefit marker-assisted breeding of Qinchuan cattle.

Signal transducer and activator of transcription 3 (STAT3) promoter methylation and expression in pituitary adenoma

BackgroundPituitary adenoma (PA) is a benign brain tumor that can cause neurological, endocrinological and ophthalmological aberrations. Till now there is a need to identify factors that can influence the tumor invasiveness and recurrence. The aim of this study was to evaluate the associations between the signal transducer and activator of transcription 3 (STAT3) promoter methylation, mRNA expression and the invasiveness or recurrence of PAs and patient clinical characteristics.MethodsStudy participants comprised of 102 subjects with a diagnosis of PA: 54 functioning and 48 non-functioning, 58 invasive and 30 non-invasive PAs and 14 relapses. The bisulfite treatment of tumor DNA and methylation-specific polymerase chain reaction (MS-PCR) method was used to determine the STAT3 gene promoter methylation. For the STAT3 mRNA expression, the first-strand cDNA was produced from total RNA by using reverse transcriptase and quantitative real-time PCR (qRT-PCR) was performed.ResultsIn 10.78% (11/102) of PA tissues STAT3 gene promoter was methylated. A gender of male and patient group older than 60 years were significantly associated with reduced STAT3 mRNA expression (Mann-Whitney test, p = 0.025, p = 0.047, respectively). However, no more statistical differences were found between STAT3 promoter methylation, mRNA expression and patient clinical characteristics or PA invasiveness or recurrence.ConclusionsFurther investigations are needed to clarify the influence of STAT3 gene promoter methylation and mRNA expression changes in PAs.

Transcriptional regulation of STAT3 by SPTBN1 and SMAD3 in HCC through cAMP-response element-binding proteins ATF3 and CREB2.

The cytoskeletal protein Spectrin, beta, non-erythrocytic 1 (SPTBN1), an adapter protein to SMAD3 in TGF-β signaling, may prevent hepatocellular carcinoma (HCC) development by downregulating the expression of signal transducer and activator of transcription 3 (STAT3). To elucidate the as yet undefined mechanisms that regulate this process, we demonstrate that higher levels of STAT3 transcription are found in livers of heterozygous SPTBN1(+/-) mice as compared to that of wild type mice. We also found increased levels of STAT3 mRNA, STAT3 protein, and p-STAT3 in human HCC cell-lines after knockdown of SPTBN1 or SMAD3, which promoted cell colony formation. Inhibition of STAT3 overrode the increase in cell colony formation due to knockdown of SPTBN1 or SMAD3. We also found that inhibition of SPTBN1 or SMAD3 upregulated STAT3 promoter activity in HCC cell-lines, which is dependent upon the cAMP-response element (CRE) and STAT-binding element (SBE) sites of the STAT3 promoter. Mechanistically, suppression of SPTBN1 and SMAD3 augmented the transcription of STAT3 by upregulating the CRE-binding proteins ATF3 and CREB2 and augmented the binding of those proteins to the regions within or upstream of the CRE site of the STAT3 promoter. Finally, in human HCC tissues, SPTBN1 expression correlated negatively with expression levels of STAT3, ATF3, and CREB2; SMAD3 expression correlated negatively with STAT3 expression; and the level of phosphorylated SMAD3 (p-SMAD3) correlated negatively with ATF3 and CREB2 protein levels. SPTBN1 and SMAD3 collaborate with CRE-binding transcription factors to inhibit STAT3, thereby preventing HCC development.

Activation of the Notch1/STAT3/Twist signaling axis promotes gastric cancer progression

Gastric carcinoma is one of the most common malignancies and a lethal cancer in the world. Notch signaling and transcription factors STAT3 (signal transducer and activator of transcription 3) and Twist regulate tumor development and are critical regulators of gastric cancer progression. Herein, the relationship among Notch, STAT3 and Twist pathways in the control of gastric cancer progression was studied. We found that Twist and phosphorylated STAT3 levels were promoted by the activated Notch1 receptor in human stomach adenocarcinoma SC-M1, embryonic kidney HEK293 and erythroleukemia K562 cells. Notch1 signaling dramatically induced Twist promoter activity through a C promoter binding factor-1-independent manner and STAT3 phosphorylation. Overexpression of Notch1 receptor intracellular domain (N1IC) enhanced the interaction between nuclear STAT3 and Twist promoter in cells. Gastric cancer progression of SC-M1 cells was promoted by N1IC through STAT3 phosphorylation and Twist expression including colony formation, migration and invasion. STAT3 regulated gastric cancer progression of SC-M1 cells via Twist. N1IC also elevated the progression of other gastric cancer cells such as AGS and KATO III cells through STAT3 and Twist. The N1IC-promoted tumor growth and lung metastasis of SC-M1 cells in mice were suppressed by the STAT3 inhibitor JSI-124 and Twist knockdown. Furthermore, Notch1 and Notch ligand Jagged1 expressions were significantly associated with phosphorylated STAT3 and Twist levels in gastric cancer tissues of patients. Taken together, these results suggest that Notch1/STAT3/Twist signaling axis is involved in progression of human gastric cancer and modulation of this cascade has potential for the targeted combination therapy.

The High-Mobility Group A1a/Signal Transducer and Activator of Transcription-3 Axis: An Achilles Heel for Hematopoietic Malignancies?

Although HMGA1 (high-mobility group A1; formerly HMG-I/Y) is an oncogene that is widely overexpressed in aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only beginning to emerge. HMGA1 encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. To determine how HMGA1 leads to neoplastic transformation, we looked for genes regulated by HMGA1 using gene expression profile analysis. Here, we show that the STAT3 gene, which encodes the signaling molecule signal transducer and activator of transcription 3 (STAT3), is a critical downstream target of HMGA1a. STAT3 mRNA and protein are up-regulated in fibroblasts overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a in fibroblasts. HMGA1a also binds directly to a conserved region of the STAT3 promoter in vivo in human leukemia cells by chromatin immunoprecipitation and activates transcription of the STAT3 promoter in transfection experiments. To determine if this pathway contributes to HMGA1-mediated transformation, we investigated STAT3 expression in our HMGA1a transgenic mice, all of which developed aggressive lymphoid malignancy. STAT3 expression was increased in the leukemia cells from our transgenics but not in control cells. Blocking STAT3 function induced apoptosis in the transgenic leukemia cells but not in controls. In primary human leukemia samples, there was a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function in human leukemia or lymphoma cells led to decreased cellular motility and foci formation. Our results show that the HMGA1a-STAT3 axis is a potential Achilles heel that could be exploited therapeutically in hematopoietic and other malignancies overexpressing HMGA1a.

The HMGA1a-STAT3 axis: an “Achilles Heel” for Hematopoietic Malignancies Overexpressing HMGA1a?

Although the high mobility group A1 (HMGA1) oncogene is widely overexpressed in high-risk hematopoietic malignancies and other aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only beginning to emerge. The HMGA1 gene encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. We showed that HMGA1 induces leukemic transformation in cultured human lymphoid cells. Inhibiting HMGA1 expression blocks the transformed phenotype in cultured human leukemia and lymphoma cells. We also engineered HMGA1a transgenic mice and all mice develop aggressive lymphoid malignancy which closely models human T-cell acute lymphoblastic leukemia. Because HMGA1 participates in transcriptional regulation, we hypothesize that it drives leukemic transformation by dysregulating specific molecular pathways. To discover genes targeted by HMGA1 in leukemic transformation, we performed gene expression profile analysis. The signaltransducer andactivator oftranscription 3 (STAT3) gene was identified as a critical downstream target of HMGA1. STAT3 mRNA and protein are up-regulated in leukemic cells overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a. HMGA1a binds directly to a conserved region of the STAT3 promoter in vivo and activates transcription of the STAT3 promoter in human leukemia cells. Blocking STAT3 function with a small molecule, platinum compound inhibitor (CPA-7) induces apoptosis in leukemic cells from HMGA1 transgenic mice, but not in control cells. In primary, human leukemia samples, there is a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function with a dominant-negative construct in human leukemia or lymphoma cells leads to decreased cellular motility and colony formation. We also showed that treatment with a small molecule, oligonucleotide inhibitor decreases the leukemic burden in the HMGA1a transgenic mice. Our results demonstrate that the HMGA1a-STAT3 axis is a potential “Achilles heel” that could be exploited therapeutically in selected hematopoietic malignancies.

STAT3 single-nucleotide polymorphisms and STAT3 mutations associated with hyper-IgE syndrome are not responsible for increased serum IgE serum levels in asthma families

Mutations in STAT3 (signal transducer and activator of transcription 3) have recently been found to cause the hyper-IgE syndrome (HIES) - a rare immunodeficiency syndrome including complex somatic features. We now tested whether STAT3 mutations or single-nucleotide polymorphisms (SNPs) within STAT3 may be responsible for increased IgE levels in asthmatic children. We genotyped DNA samples from 918 individuals of 217 core families by MALDI-TOF mass spectrometry. SNPs were selected from previous reports, by functional relevance and haplotype-tagging capacity. In 24 assays, including the recently described HIES mutations, no variant was detected. In another 27 SNP assays, there was no association of any STAT3 variant with asthma, allergic rhinitis or eczema. In addition, neither total and specific IgE and eosinophil count nor any lung function parameter showed any significant association. When combining high eosinophil counts and high total IgE levels to an HIES-like trait, four SNPs in the 5'-UTR of STAT3 were slightly overtransmitted. A minor fraction of asthmatic children may possibly have an alternate STAT3 promoter architecture influencing joined IgE and eosinophil upregulation. While an overall effect of STAT3 mutations on serum IgE is unlikely in asthma children.

Integration of HIV-1 caused STAT3-associated B cell lymphoma in an AIDS patient

Signal transducer and activator of transcription 3 (STAT3) is a DNA-binding transcription factor activated by multiple cytokines and interferons. High expression of STAT3 has also been implicated in cancer and lymphoma. Here, we show a case of B cell lymphoma in which a defective human immunodeficiency virus 1 (HIV-1) integrated upstream of the first STAT3 coding exon. The lymphoma cells with anaplastic large cell morphology formed multiple nodular lesions in the lung of an acquired immunodeficiency syndrome (AIDS) patient with Kaposi's sarcoma. The provirus had a 5′ long terminal repeat (LTR) deletion, but the 3′ LTR had stronger promoter activity than the STAT3 promoter in reporter assays. Immunohistochemistry showed increased expression of STAT3 in the nuclei of lymphoma cells. Transfection of STAT3 resulted in transient cell proliferation in primary B cells in vitro. Although this is a very rare case of HIV-1-integrated lymphoma, these data suggest that up-regulation of STAT3 caused by HIV-1 integration resulted in the development of B cell lymphoma in this special case.