Expression Of STAT3 Target Genes Research Articles

HIC1 attenuates invasion and metastasis by inhibiting the IL-6/STAT3 signalling pathway in human pancreatic cancer

Hypermethylated in cancer 1 (HIC1) is a tumour suppressor gene that is frequently deleted or epigenetically silenced in many human cancers. However, the molecular function of HIC1 in pancreatic cancer has not been fully elucidated, especially in cancer invasion and metastasis. We aimed to clarify the clinical relevance of HIC1 and human pancreatic cancer and the mechanism of its effect on invasion and metastasis .HIC1 was downregulated in pancreatic cancer patient cancer tissue and pancreatic cancer cell lines. A tissue microarray analysis demonstrated that negative HIC1 expression predicted advanced pathological stages and worse patient survival. In addition, HIC1 inhibited the invasion and metastasis of pancreatic cancer cells both in vitro and in vivo. Finally, HIC1 repressed the expression of STAT3 target genes, including c-Myc, VEGF, CyclinD1, MMP2 and MMP9, by binding and interacting with STAT3 to impede its DNA-binding ability but without affecting the protein levels of STAT3 and p-STAT3. Therefore, HIC1 appears to function as a STAT3 inhibitor and may be a promising target for cancer research and for the development of an optimal treatment approach for pancreatic cancer.

Activating Mutation of STAT3 Protects Lymphocytes from Apoptosis and Leads to a Clinical Phenotype Resembling the Autoimmune Lymphoproliferative Syndrome

(PS and UF contributed equally to this work.)Introduction:The Autoimmune Lymphoproliferative Syndrome (ALPS) is caused by inefficient clearing of T lymphocytes. Patients are thus characterized by lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias and an elevated number of double negative T cells (CD3+, TCRα/β+, CD4-, CD8-). Patients suffering from ALPS typically harbor germline or somatic mutations in genes involved in the apoptotic FAS death receptor signaling pathway (FAS, FASLG or CASP10). For 20-30% of patients, however, the genetic cause is still unknown.Methods:The objective of this study was to identify novel gene candidates underlying ALPS of unknown genetic cause. To this end, 25 patients with clinical ALPS symptoms, but without classical mutations were analyzed by whole-exome sequencing. The list of potential candidates was narrowed down using an in-house developed bioinformatic analysis pipeline for patient-based gene prioritization based on protein-protein interaction networks. Resulting candidates were validated by Sanger sequencing and their impact on Fas signaling was studied.ResultsWe identified a de novo germline mutation of the Signal Transducer And Activator Of Transcription 3 (STAT3, c.833G>A, p.R278H) in one of the analyzed patients. The patient presented at the age of nine with Coombs positive hemolytic anemia, thrombocytopenia, generalized progressive, non-infectious, non-malignant lymphadenopathy and splenomegaly. Immunophenotyping revealed increased numbers of double negative T cells (20% in peripheral blood) and over time the patient developed panhypogammaglobulinemia.We performed immunoblot analyses and could demonstrate that the level of phosphorylated STAT3 (pSTAT3-Tyr705) was elevated in the patient's lymphocytes. This finding indicated that the mutation leads to constitutive activation of STAT3. Consistently, we detected an increased expression of STAT3 target genes (including SOCS3, MMP7 and the anti-apoptotic factors BCL2 and BCL2L1) compared to wild-type controls using quantitative real-time PCR. We could also show a decreased expression of the pro-apoptotic genes BAK1 and BAX that is in accordance with the known negative regulation by STAT3. Thus, in the analyzed patient we found that the balance of pro- and anti-apoptotic factors inside the cell was skewed towards apoptosis resistance.Consistently, we could induce apoptosis in vitro applying recombinant Fas ligand, IL21 or staurosporine efficiently in cells derived from healthy controls, but only to a significantly lesser extent in cells from the patient. Moreover, in healthy cells we observed a concurrent downregulation of anti-apoptotic BCL2/BCL2L1 and an upregulation of pro-apoptotic BAX/BAK1 expression upon treatment that was completely absent in the patient's cells.Next, we tried to rescue the effect of constitutively activated STAT3 by application of a STAT3 specific inhibitor: S3I-201. When we treated the patient's lymphocytes with S3I-201 the expression levels of pro- and anti-apoptotic genes were similar to healthy controls and the sensitivity to apoptosis was restored.Conclusion:We report here a novel germline dominant STAT3 gain-of-function mutation that caused a clinical phenotype mimicking ALPS. Recent studies indicated that dominant germline STAT3 gain-of-function mutations lead to autoimmunity, hypogammaglobulinemia, and lymphoproliferation. STAT3 gain-of-function patients therefore share some clinical characteristics with ALPS patients. The clinical presentation of the patient described here differed from the phenotypes previously reported and thus extends the spectrum of STAT3 -associated diseases. The mechanism underlying the clinical symptoms of STAT3 gain-of-function patients has not yet been determined. Here, we demonstrate increased activation of STAT3 and STAT3 target genes, leading to a skewed balance of pro- and anti-apoptotic factors and apoptosis evasion as a cause for lymphocyte accumulation and resulting autoimmunity in a STAT3 gain-of-function patient. Similar to ALPS patients, diminished responsiveness of lymphocytes to apoptosis seems to be a major characteristic. The clinical phenotype may differ because mutations in STAT3 or Fas signaling genes, respectively, affect overlapping, but also distinct signaling pathways. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic regulation and anti-tumorigenic effects of SH2-containing protein tyrosine phosphatase 1 (SHP1) in human gastric cancer cells.

SH2-containing protein tyrosine phosphatase 1 (SHP1) is an important negative regulator in cytokine-mediated signal transduction and cell cycling. Recent studies have demonstrated that SHP1 promoter methylation is frequently observed in gastric adenocarcinoma tissues. In this in vitro study, we attempted to reveal promoter hypermethylation and to investigate effects of SHP1 in gastric carcinoma cell lines. We observed that both gene and protein expression of SHP1 were negative in 8 of 10 gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-638, SNU-719, MKN-28, MKN-45, AGS). Methylation-specific PCR (MSP) showed a methylation-specific band only in the 10 gastric cancer lines. Bisulfite pyrosequencing in AGS, MKN-28, and SNU-719 cells indicated that methylation frequency was as high as 94.4, 92.6, and 94.5%, respectively, in the three cell lines. Treatment of SNU-719, MKN-28, and AGS cells with 5-Aza-2'-deoxycytidine (5-Aza-dc) led to re-expression of SHP1 in these cells. Introduction of exogenous SHP1 in SNU-719 and MKN-28 cells by transient transfection substantially downregulated protein expression of constitutive phosphor-Janus kinase 2 (JAK2) (tyrosine 1007/1008) and phosphor-signal transducers and activators of transcription 3 (STAT3) (tyrosine 705), which in turn decreased expression of STAT3 target genes including those encoding cyclin D1, MMP-9, VEGF-1, and survivin. Induction of SHP1 significantly inhibited cell proliferation, migration and invasion in SNU-719 and MKN-28 cells. Taken together, epigenetic silencing of SHP1 is frequently caused by promoter hypermethylation in gastric carcinoma cells. Overexpression of SHP1 downregulates the JAK2/STAT3 pathway to modulate various target genes and inhibit cell proliferation, migration, and invasion in gastric cancer cells.

Abstract A25: Dysregulation of MYC via STAT1 promotes tumor progression in serous papillary endometrial cancer

Abstract Serous papillary endometrial cancers (SPEC) harboring several genetic alterations, such as TP53 mutation, are known highly progressive with worse prognosis compared with endometrioid counter-part, but it still remains unclear which molecular pathway is responsible for their unfavorable features. The IFN-γ; transcription factor signal transducer and activator of transcription 1 (STAT1) has been considered as a tumor suppressor with transcription-dependent and transcription-independent mechanisms, but recent studies showed STAT1 was associated with poor prognosis, and was involved in maintaining basal expression of tumor pro-survival STAT3 target genes, such as MYC, with unclear mechanism in some cancers. A previous report of the cancer genome atlas (TCGA) indicated MYC amplification in some SPECs; as MYC is known to be maintained also by STAT1, we investigate the role of STAT1-MYC axis in the malignant features of SPECs. Gene expression microarray was conducted for 63 endometrial cancer samples and SPEC cell line, SPAC-1L. Immunohistochemical staining were conducted using endometrial cancer samples under protocols approved by the Institutional Review Board to investigate SPEC-specific pathways (Kyoto cohort: n=91, Vancouver cohort: n=462). Using SPAC-1L cell line, several functional assay in vitro such as cellular proliferation, migration, invasion and apoptosis under treatment of cisplatin, doxorubicin, and paclitaxel were assessed with or without suppressing target genes. In vivo tumorigenesis was performed in mouse model and some computational analysis were done in silico. Gene expression microarray analysis revealed STAT1 pathway was commonly activated in SPECs both in our dataset and TCGA dataset as a key molecule of ‘SPEC signature’. In two independent cohorts, STAT1 expression was confirmed significantly higher in SPECs (p<0.001), and the disease specific survival of EC with high STAT1 expression was worse than those with low STAT1 expression (p<0.05). We found that STAT1 regulates MYC as well as ICAM1, PD-L1, and SMAD7, as well as the capacity for proliferation, adhesion, migration, invasion, and in vivo tumorigenecity in cells with a high SPEC signature. In contrast, suppression of STAT1 attenuated induction of these genes and inhibited xenograft tumor growth on NOD-SCID mice. Furthermore, suppressing STAT1 expression by shRNA sensitized SPEC cells to cisplatin. In STAT1 suppressed cells, apoptosis and cleaved caspase3 expression was confirmed even under low dose cisplatin treatment (p<0.05). Based on silico analysis using cBioPortal, we found amplification of MYC and up-regulated of MYC mRNA in 42% SPECs. The 227 up-regulated genes in the SPEC signature harbor the MYC binding site motif by GATHER analysis (p<0.001), and the predictive MYC activity signature score was statistically higher in SPECs than in other subtypes of endometrial cancers, while that in SPAC-1L cells was diminished with STAT1 knockdown. These results indicate that STAT1 pathway modulates the micro-environment of SPECs in deregulating MYC, as well as other target genes, to promote tumor progression. Furthermore, STAT1-MYC axis could be developed as a novel potential target for molecular targeting therapy in SPECs. Citation Format: Budiman Kharma, Tsukasa Baba, Noriomi Matsumura, Ryusuke Murakami, Ken Yamaguchi, Junzo Hamanishi, Masaki Mandai, Ikuo Konishi. Dysregulation of MYC via STAT1 promotes tumor progression in serous papillary endometrial cancer. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A25.

Impact of the N-Terminal Domain of STAT3 in STAT3-Dependent Transcriptional Activity.

The transcription factor STAT3 is constitutively active in many cancers, where it mediates important biological effects, including cell proliferation, differentiation, survival, and angiogenesis. The N-terminal domain (NTD) of STAT3 performs multiple functions, such as cooperative DNA binding, nuclear translocation, and protein-protein interactions. However, it is unclear which subsets of STAT3 target genes depend on the NTD for transcriptional regulation. To identify such genes, we compared gene expression in STAT3-null mouse embryonic fibroblasts (MEFs) stably expressing wild-type STAT3 or STAT3 from which NTD was deleted. NTD deletion reduced the cytokine-induced expression of specific STAT3 target genes by decreasing STAT3 binding to their regulatory regions. To better understand the potential mechanisms of this effect, we determined the crystal structure of the STAT3 NTD and identified a dimer interface responsible for cooperative DNA binding in vitro. We also observed an Ni(2+)-mediated oligomer with an as yet unknown biological function. Mutations on both dimer and Ni(2+)-mediated interfaces affected the cytokine induction of STAT3 target genes. These studies shed light on the role of the NTD in transcriptional regulation by STAT3 and provide a structural template with which to design STAT3 NTD inhibitors with potential therapeutic value.

Niclosamide suppresses cell migration and invasion in enzalutamide resistant prostate cancer cells via Stat3-AR axis inhibition.

It is known that over expression of IL6 in prostate cancer cells confer enzalutamide resistance and that this may occur through constitutive Stat3 activation. Additionally, recent pre-clinical studies suggested enzalutamide might have the potential adverse effect of inducing metastasis of prostate cancer cells via Stat3 activation. This study is aimed to target Stat3 activation and improve enzalutamide therapy. Sensitivity of prostate cancer cells to enzalutamide was tested using cell growth assays and clonogenic assays. Wound healing and invasion assays were performed to determine cell migration and invasion in vitro. Quantitative reverse transcription-PCR, ELISA and Western blotting were performed to detect expression levels of PSA, c-Myc, survivin, Stat3, and AR. ChIP assay was performed to examine recruitment of AR to the PSA promoter. In the present study, we found niclosamide, a previously identified novel inhibitor of androgen receptor variant (AR-V7), inhibited Stat3 phosphorylation, and expression of downstream target genes. Niclosamide synergistically reversed enzalutamide resistance in prostate cancer cells and combination treatment of niclosamide with enzalutamide significantly induced cell apoptosis and inhibited cell growth, colony formation, cell migration and invasion. Knock down of Stat3 abrogated enzalutamide resistance resulting in reduced recruitment of AR to the PSA promoter in prostate cancer cells expressing IL6. Moreover, niclosamide reversed enzalutamide resistance by down-regulating Stat3 target gene expression Stat3and abrogating recruitment of AR to PSA promoter resulting in PSA inhibition. This study demonstrated the IL6-Stat3-AR axis in prostate cancer is one of the crucial mechanisms of enzalutamide resistance. Niclosamide has the potential to target the IL6-Stat3-AR pathway to overcome enzalutamide resistance and inhibit migration and invasion in advanced prostate cancer.

Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function.

Signal transducer and activator of transcription STAT5 is essential for the regulation of proliferation and survival genes. Its activity is tightly regulated through cytokine signaling and is often upregulated in cancer. We showed previously that the deacetylase inhibitor trichostatin A (TSA) inhibits STAT5-mediated transcription by preventing recruitment of the transcriptional machinery at a step following STAT5 binding to DNA. The mechanism and factors involved in this inhibition remain unknown. We now show that deacetylase inhibitors do not target STAT5 acetylation, as we initially hypothesized. Instead, they induce a rapid increase in global histone acetylation apparently resulting in the delocalization of the bromodomain and extra-terminal (BET) protein Brd2 and of the Brd2-associated factor TBP to hyperacetylated chromatin. Treatment with the BET inhibitor (+)-JQ1 inhibited expression of STAT5 target genes, supporting a role of BET proteins in the regulation of STAT5 activity. Accordingly, chromatin immunoprecipitation demonstrated that Brd2 is associated with the transcriptionally active STAT5 target gene Cis and is displaced upon TSA treatment. Our data therefore indicate that Brd2 is required for the proper recruitment of the transcriptional machinery at STAT5 target genes and that deacetylase inhibitors suppress STAT5-mediated transcription by interfering with Brd2 function.

Artocarpus altilis (Parkinson) Fosberg Extracts and Geranyl Dihydrochalcone Inhibit STAT3 Activity in Prostate Cancer DU145 Cells.

Artocarpus altilis (Parkinson) Fosberg has traditionally been used in Indonesia for the treatment of liver cirrhosis, hypertension, and diabetes. In many other countries, it is used for the treatment of malaria, yellow fever, and dengue fever. It has been reported that A. altilis extracts have antiatherosclerotic and cytoprotective effects, but its molecular targets in tumor cells are not yet fully understood. The A. altilis extracts and the partially purified fraction have been shown to inhibit STAT3 activity and the phosphorylation of STAT3 in a dose-dependent manner. To identify the active components, a bioassay-guided isolation of the partially purified fraction resulted in the identification of a geranyl dihydrochalcone, CG901. Its chemical structure was established on the basis of spectroscopic evidence and comparison with published data. The partially purified fraction and the isolated a geranyl dihydrochalcone, CG901, down-regulated the expression of STAT3 target genes, induced apoptosis in DU145 prostate cancer cells via caspase-3 and PARP degradation, and inhibited tumor growth in human prostate tumor (DU145) xenograft initiation model. These results suggest that A. altilis could be a good natural source and that the isolated compound will be a potential lead molecule for developing novel therapeutics against STAT3-related diseases, including cancer and inflammation.

Dysregulation of IL-17 Production in Aged T-Lymphocytes Promotes Pro-tumorigenic Changes in Prostate Epithelial Cells

Event Abstract Back to Event Dysregulation of IL-17 Production in Aged T-Lymphocytes Promotes Pro-tumorigenic Changes in Prostate Epithelial Cells Alejandra De Angulo1*, Robert Faris1, Christopher Jolly1 and Linda DeGraffenried1 1 University of Texas at Austin, United States Background: Prostate cancer represents 8% of all new cancer cases world-wide and is the second leading cause of cancer death in men. Incidence is closely associated with aging, but the mechanisms by which aging promotes prostate cancer development are unclear, limiting the development of effective preventive interventions. Aging is accompanied by immune dysfunction and a progressive increase in pro-inflammatory cytokines, including Interleukin 17 (IL-17). Aging-associated increases in circulating IL-17 promotes pro-inflammatory signaling in prostate epithelial cells, possibly contributing to tumorigenesis. Methods: Serum and splenic CD4+ T-lymphocytes from young T cell aging-mimic mice as well as young and aged wild-type mice were collected. Surface markers and intracellular levels of IL- 17, IFN-γ and IL-4 in isolated CD4+ T-cells were measured using flow-cytometry. shRNA was used to knock down the IL-17 receptor in LNCaP prostate cancer cells and RWPE-1 non-transformed prostate epithelial cells, which were then exposed to mouse sera or conditioned media from stimulated T-lymphocytes. NF-kB and STAT3 activation, NF-kB and STAT3 target gene expression, and epithelial cell transformation were all measured in prostate epithelial cells. Results: T cells from aging and aging-mimic mice secrete elevated levels of IL-17, possibly due to an imbalance in the TH17/TH1 and TH17/TH2 cell ratios. T-lymphocyte-secreted IL-17 from aging-mimic mice induced NF-kB and STAT3 activity and target gene expression in LNCaP and RWPE-1 cells. Importantly, IL-17 also promoted pro-tumorigenic changes in RWPE-1 cells, leading to a pre-transformed phenotype. Inhibition of IL-17 signaling blocked age-induced changes in both the cancer and non-transformed prostate epithelial cells. Conclusion: Aging of the immune system is associated with increased levels of IL-17, which stimulates the pro-inflammatory NF-kB and STAT3 pathways in prostate epithelial cells and activates programming associated with cellular transformation. Inhibition of IL-17 signaling blocks the pro-tumorigenic activity induced by aging T cells. These findings provide evidence that the dysregulation of IL-17 production in aged T-cells may directly contribute to the increased risk for prostate cancer. Future novel immunotherapies for prostate cancer prevention could target the increase production of IL-17 associated with aging

Abstract B51: SOCS3 deficiency in myeloid cells promotes prostate tumor development

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cell leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributes of SOCS3. Citation Format: Hao Yu, Yudong Liu, Jessy Deshane, Etty (Tika) Benveniste, Hongwei Qin. SOCS3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B51. doi:10.1158/1538-7445.CHTME14-B51

Abstract A25: SOCS3 deficiency in myeloid cells promotes prostate tumor development

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cells leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributer of SOCS3. Citation Format: Hao Yu, Yudong Liu, Braden C. McFarland, Etty N. Benveniste, Hongwei Qin. SOCS3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A25. doi:10.1158/1538-7445.CHTME14-A25

Stat5 gene dosage in T cells modulates CD8+ T-cell homeostasis and attenuates contact hypersensitivity response in mice.

Contact hypersensitivity assay (CHS) faithfully models human allergies. The Stat5 transcription factors are essential for both lymphocyte development and acute immune responses. Although consequences of Stat5 ablation and transgenic overexpression for the lymphocyte development and functions have been extensively studied, the role of Stat5 gene dosage in contact allergies has not been addressed. We investigated the effect of Stat5 gene dosage modulation in contact allergies using CHS in mice. Transgenic animals heterozygous for the germline Stat5 null allele were subjected to CHS. To dissect cell type sensitive to Stat5 gene dosage, animals with Stat5 haplo-insufficiency in T cells, where one Stat5 allele was removed by Lck-Cre-mediated deletion (Stat5(ΔT/+)), were tested by CHS. Frequency of T cells, B cells, and monocytes were analyzed in Stat5(ΔT/+) and wild-type animals by flow cytometry. Proliferation of Stat5(ΔT/+) CD8(+) T cells was studied in vitro by stimulation with IL-4 and IL-2 cytokines, and changes in the expression of Stat5 target genes were assayed by quantitative real-time PCR assay. Haplo-insufficiency of Stat5 in T cells leads to the reduction in CD8(+) T cells in all lymphoid organs and attenuates CHS response. Stat5(ΔT/+) CD8(+) T cells failed to fully activate Stat5-dependent expression of cell cycle/survival target genes, such as Bcl2 and Pim1, and to proliferate efficiently in response to IL-2 and IL-4 cytokine. Our data identify Stat5 as a dose-dependent regulator of CD8(+) T-cell functions in contact allergies and suggest that modulation of Stat5 dosage could be used to target contact allergies in humans.

Metformin enhances the anti-adipogenic effects of atorvastatin via modulation of STAT3 and TGF-β/Smad3 signaling

Adipocyte accumulation is associated with the development of obesity and obesity-related diseases. Interactions of master transcription factors and signaling cascades are required for adipogenesis. Regulation of excessive adipogenic processes may be an attractive therapeutic for treatment of obesity and obesity-related diseases. In this study, we found that atorvastatin exerts an anti-adipogenic activity in 3T3-L1 pre-adipocytes, and that this activity is elevated in combination with metformin. Expression of the adipogenic master regulators PPARγ and C/EBPα, and their target gene aP2, was suppressed by atorvastatin. Furthermore, atorvastatin treatment resulted in increased activation of the key master regulator of cellular energy homeostasis, AMPK. These biological activities of atorvastatin were elevated in combination with metformin. These anti-adipogenic activities were associated with regulation of the STAT3 and TGF-β signaling cascades, resulting in the regulation of the expression of STAT3 target genes, such as KLF5, p53, and cyclin D1, and TGF-β signaling inhibitory genes, such as SMAD7. Our results suggest that combination therapy with atorvastatin and metformin may have therapeutic potential for the treatment of obesity and obesity-related diseases caused by excessive adipogenesis.

Astaxanthin inhibits JAK/STAT-3 signaling to abrogate cell proliferation, invasion and angiogenesis in a hamster model of oral cancer.

Identifying agents that inhibit STAT-3, a cytosolic transcription factor involved in the activation of various genes implicated in tumour progression is a promising strategy for cancer chemoprevention. In the present study, we investigated the effect of dietary astaxanthin on JAK-2/STAT-3 signaling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by examining the mRNA and protein expression of JAK/STAT-3 and its target genes. Quantitative RT-PCR, immunoblotting and immunohistochemical analyses revealed that astaxanthin supplementation inhibits key events in JAK/STAT signaling especially STAT-3 phosphorylation and subsequent nuclear translocation of STAT-3. Furthermore, astaxanthin downregulated the expression of STAT-3 target genes involved in cell proliferation, invasion and angiogenesis, and reduced microvascular density, thereby preventing tumour progression. Molecular docking analysis confirmed inhibitory effects of astaxanthin on STAT signaling and angiogenesis. Cell culture experiments with the endothelial cell line ECV304 substantiated the role of astaxanthin in suppressing angiogenesis. Taken together, our data provide substantial evidence that dietary astaxanthin prevents the development and progression of HBP carcinomas through the inhibition of JAK-2/STAT-3 signaling and its downstream events. Thus, astaxanthin that functions as a potent inhibitor of tumour development and progression by targeting JAK/STAT signaling may be an ideal candidate for cancer chemoprevention.

Abstract 1068: Socs3 deficiency in myeloid cells promotes prostate tumor development

Abstract Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and have been implicated as functioning as tumor suppressors, particularly SOCS3. We have previously demonstrated that specific deletion of SOCS3 in myeloid cells leads to heightened activation of STAT3 and downstream STAT3 target gene expression. STAT3 activation has been strongly implicated in contributing to prostate cancer progression and metastasis. STAT3 signaling in myeloid cells has been demonstrated to attenuate anti-tumor immunity. Thus, we evaluated the impact of SOCS3 deletion in myeloid cells in a murine prostate tumor model. We demonstrate that prostate tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Myeloid Derived Suppressor Cells (MDSC) in both the spleen and tumor microenvironment, and less CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived mononuclear cells exhibit heightened STAT3 activation and are preferentially differentiate into MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the prostate tumor microenvironment that promotes development of Gr-1+CD11b+ cells via a STAT3-depentent pathway. Abrogation of tumor-derived G-CSF reduced the proliferation and accumulation of Gr-1+CD11b+ cells and inhibited prostate tumor growth. Therefore, the tumor-derived cytokine, G-CSF, is a prospective candidate upstream of the STAT3/SOCS3 pathway in MDSC differentiation from the bone marrow. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, which may contribute to tumor suppressor attributer of SOCS3. Citation Format: Hao Yu, Hongwei Qin. Socs3 deficiency in myeloid cells promotes prostate tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1068. doi:10.1158/1538-7445.AM2014-1068

The natural chemopreventive agent sulforaphane inhibits STAT5 activity.

Signal transducer and activator of transcription STAT5 is an essential mediator of cytokine, growth factor and hormone signaling. While its activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated to a number of hematological and solid tumor cancers. We previously showed that deacetylase inhibitors can inhibit STAT5 transcriptional activity. We now investigated whether the dietary chemopreventive agent sulforaphane, known for its activity as deacetylase inhibitor, might also inhibit STAT5 activity and thus could act as a chemopreventive agent in STAT5-associated cancers. We describe here sulforaphane (SFN) as a novel STAT5 inhibitor. We showed that SFN, like the deacetylase inhibitor trichostatin A (TSA), can inhibit expression of STAT5 target genes in the B cell line Ba/F3, as well as in its transformed counterpart Ba/F3-1*6 and in the human leukemic cell line K562 both of which express a constitutively active form of STAT5. Similarly to TSA, SFN does not alter STAT5 initial activation by phosphorylation or binding to the promoter of specific target genes, in favor of a downstream transcriptional inhibitory effect. Chromatin immunoprecipitation assays revealed that, in contrast to TSA however, SFN only partially impaired the recruitment of RNA polymerase II at STAT5 target genes and did not alter histone H3 and H4 acetylation, suggesting an inhibitory mechanism distinct from that of TSA. Altogether, our data revealed that the natural compound sulforaphane can inhibit STAT5 downstream activity, and as such represents an attractive cancer chemoprotective agent targeting the STAT5 signaling pathway.

“Activated” STAT Proteins: A Paradoxical Consequence of Inhibited JAK-STAT Signaling in Cytomegalovirus-Infected Cells

We have previously characterized mouse CMV (MCMV)-encoded immune-evasive IFN signaling inhibition and identified the viral protein pM27 as inducer of proteasomal degradation of STAT2. Extending our analysis to STAT1 and STAT3, we found that MCMV infection neither destabilizes STAT1 protein nor prevents STAT1 tyrosine Y701 phosphorylation, nuclear translocation, or the capability to bind γ-activated sequence DNA-enhancer elements. Unexpectedly, the analysis of STAT3 revealed an induction of STAT3 Y705 phosphorylation by MCMV. In parallel, we found decreasing STAT3 protein amounts upon MCMV infection, although STAT3 expression normally is positive autoregulative. STAT3 phosphorylation depended on the duration of MCMV infection, the infectious dose, and MCMV gene expression but was independent of IFNAR1, IL-10, IL-6, and JAK2. Although STAT3 phosphorylation did not require MCMV immediate early 1, pM27, and late gene expression, it was restricted to MCMV-infected cells and not transmitted to bystander cells. Despite intact STAT1 Y701 phosphorylation, IFN-γ-induced target gene transcription (e.g., IRF1 and suppressor of cytokine signaling [SOCS] 1) was strongly impaired. Likewise, the induction of STAT3 target genes (e.g., SOCS3) by IL-6 was also abolished, indicating that MCMV antagonizes STAT1 and STAT3 despite the occurrence of tyrosine phosphorylation. Consistent with the lack of SOCS1 induction, STAT1 phosphorylation was prolonged upon IFN-γ treatment. We conclude that the inhibition of canonical STAT1 and STAT3 target gene expression abrogates their intrinsic negative feedback loops, leading to accumulation of phospho-tyrosine-STAT3 and prolonged STAT1 phosphorylation. These findings challenge the generalization of tyrosine-phosphorylated STATs necessarily being transcriptional active and document antagonistic effects of MCMV on STAT1/3-dependent target gene expression.

Cucurbitacin B and cucurbitacin I suppress adipocyte differentiation through inhibition of STAT3 signaling

Cucurbitacin B, a member of the cucurbitaceae family, can act as a STAT3 signaling inhibitor to regulate the growth of hepatocellular carcinoma. STAT3 signaling has been shown to inhibit adipocyte differentiation through C/EBPα and PPARγ. Based on these studies, we hypothesized that cucurbitacin B would prevent PPARγ mediated adipocyte differentiation through STAT3 signaling. To test this hypothesis, mesenchymal C3H10T1/2 and 3T3-L1 preadipocyte cells were treated with a sub-cytotoxic concentration of cucurbitacin B. Cucurbitacin B treatment inhibits lipid accumulation and expression of adipocyte markers including PPARγ and its target genes in a dose-dependent manner. Cucurbitacin B treatment impairs STAT3 signaling as manifested by reduced phosphorylation of STAT3 and suppression of STAT3 target gene expression in preadipocytes. The anti-adipogenic effects of cucurbitacin B are significantly blunted in cells with STAT3 silenced by introducing small interfering RNA. Finally, our data show that cucurbitacin I, another cucurbitacin family member, also inhibits adipocyte differentiation by suppressing STAT3 signaling. Together, our data suggest the possibility of utilizing cucurbitacins as a new strategy to treat metabolic diseases and implicate STAT3 as a new target for the development of functional foods and drugs.

A Novel Small Molecule STAT Inhibitor, BP-4-018, Demonstrates Significant Anti-Tumour Activity and Synergism With Bortezomib In Pre-Clinical In Vivo Models Of Multiple Myeloma

Despite significant advances in the treatment of Multiple Myeloma (MM), it remains incurable and thus, novel therapeutic strategies are critically needed. Prior work has demonstrated that the transcription factor STAT3 is aberrantly activated in a number of human tumors and drives hallmark oncogenic processes such as proliferation, survival, angiogenesis and drug resistance. As such, this master regulator of malignant processes has become the focus of therapeutic targeting in human tumors that harbor constitutive STAT3 activation, including MM. In the present studies, we characterize a newly developed small molecule, BP-4-018. The inhibitory mechanism of BP-4-018 is based on its ability to function as a phosphotyrosine mimetic, and thus bind the STAT3 SH2 domain to inhibit STAT3 phosphorylation (pSTAT3), STAT3 dimerization and DNA binding. Based on the 3D crystal structure of STAT3, SH2 docking simulations and fluorescence polarization assays, BP-4-018 demonstrates high affinity binding to the SH2 domain of STAT3. Using a panel of molecularly heterogeneous human myeloma cell lines (HMCLs), BP-4-018 demonstrated potent and broad anti-tumor activity in MTT assays, with IC50 values in the low μM range (1.91 μM - 6.48 μM). These anti-MM effects are mediated through the induction of the apoptosis as evidence by Annexin V+ staining, PARP cleavage and activation of caspases 3 and 9. Using patient derived bone marrow (BM) cells, BP-4-018 was shown to retain its anti-MM activity against HMCLs cultured in the presence of a BM stroma, induce apoptosis in primary MM tumor cells, and importantly, displayed little to no toxicity against non-malignant BM cells. Using cell-based assays, we show that BP-4-018 inhibits basal and cytokine-induced pSTAT3, as well as STAT3 transcriptional activity in HMCLs that stably express a STAT3-driven luciferase reporter construct. Consistent with these effects, western blot analyses of BP-4-018-treated cells reveal decreased expression of STAT3 target genes including c-Myc, Mcl-1 and Bcl-xL. Interestingly, BP-4-018 did not demonstrate preferential inhibition of cytokine induced STAT3 phosphorylation over that of STATs 1 or 5 suggesting that this compound may in fact be a pan-STAT inhibitor. BP-4-018 does not however inhibit the phosphorylation of other proteins whose regulation is SH2-dependent including AKT, ERK, SRC and LYN. In vitro combinatorial studies revealed that BP-4-018 is synergistic in combination with dexamethasone (CI=0.62), melphalan (CI=0.94), lenalidomide (CI=0.82) and is highly synergistic with bortezomib (BTZ) (CI=0.37). To examine the potential mechanisms of combinatorial synergy with BTZ, we evaluated the effects of BTZ on STAT3 signaling in HMCLs. BTZ treatment alone augmented STAT3 transcriptional activity and led to the accumulation of Mcl-1 protein, which was reversed when cells were co-treated with BP-4-018. Furthermore, HMCLs expressing a dominant negative STAT3 mutant display increased sensitivity to BTZ. Collectively these data suggest that the combinatorial synergism with BTZ is at least in part related to BP-4-018-mediated inhibition of STAT3. Finally, we used a novel STAT3-driven luciferase xenograft model of MM to show that BP-4-018 (15 mg/kg/day, PO) reduces STAT3-driven luciferase activity in vivo, significantly delays tumor growth as a single agent, and in combination with BTZ, induces marked and in some animals complete tumor regression. Neither BP-4-018 alone or in combination elicited significant toxicities as assessed by weight loss, and more extensive pharmacodynamic, pharmacokinetic and toxicology studies of BP-4-018 are currently underway. Taken together, our results highlight the promising therapeutic potential of BP-4-018 and support the continued development of targeted therapies for MM with a focus on aberrant STAT signaling. Disclosures: No relevant conflicts of interest to declare.

NF-κB-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma

Glioblastoma (GBM) is the most aggressive, neurologically destructive and deadly tumor of the central nervous system (CNS). In GBM, the transcription factors NF-κB and STAT3 are aberrantly activated and associated with tumor cell proliferation, survival, invasion and chemoresistance. In addition, common activators of NF-κB and STAT3, including TNF-α and IL-6, respectively, are abundantly expressed in GBM tumors. Herein, we sought to elucidate the signaling crosstalk that occurs between the NF-κB and STAT3 pathways in GBM tumors. Using cultured GBM cell lines as well as primary human GBM xenografts, we elucidated the signaling crosstalk between the NF-κB and STAT3 pathways utilizing approaches that either a) reduce NF-κB p65 expression, b) inhibit NF-κB activation, c) interfere with IL-6 signaling, or d) inhibit STAT3 activation. Using the clinically relevant human GBM xenograft model, we assessed the efficacy of inhibiting NF-κB and/or STAT3 alone or in combination in mice bearing intracranial xenograft tumors in vivo. We demonstrate that TNF-α-induced activation of NF-κB is sufficient to induce IL-6 expression, activate STAT3, and elevate STAT3 target gene expression in GBM cell lines and human GBM xenografts in vitro. Moreover, the combined inhibition of NF-κB and STAT3 signaling significantly increases survival of mice bearing intracranial tumors. We propose that in GBM, the activation of NF-κB ensures subsequent STAT3 activation through the expression of IL-6. These data verify that pharmacological interventions to effectively inhibit the activity of both NF-κB and STAT3 transcription factors must be used in order to reduce glioma size and aggressiveness.