STAT3-dependent Manner Research Articles

STAT3 Inhibitor OPB-51602 Is Cytotoxic to Tumor Cells Through Inhibition of Complex I and ROS Induction.

SummarySTAT3 is a transcription factor involved in several cellular activities including inflammation, proliferation, and survival, but it also plays a non-transcriptional role in modulating mitochondrial metabolism. Given its diverse functions in human cancers, it is an emerging therapeutic target. Here we show that OPB-51602, a small molecule inhibitor of STAT3, is highly toxic in a STAT3-dependent manner. Specifically, drug toxicity depends on mitochondrial STAT3 as tumor cells expressing only a mitochondrially restricted form of STAT3 are sensitive to the compound, whereas STAT3-null cells are protected. OPB-51602 inhibited complex I activity and led to increased ROS production, which in turn induced mitophagy, actin rearrangements, and cell death. Cells undergoing reduced oxidative phosphorylation or expressing NDI1 NADH dehydrogenase from Saccharomyces cerevisiae, which bypasses mammalian complex I, were resistant to OPB-51602 toxicity. These results show that targeting mitochondrial STAT3 function causes synthetic lethality through complex I inhibition that could be exploited for cancer chemotherapy.

Abstract 1608: Adaptive resistance to PD-1 blockade upregulates Tim-3 through STAT3 pathway in regulatory T cells in melanoma

Abstract Tregs play an opposing role in anti-tumour immunity by suppressing effective immunotherapies in melanoma. Thus, understanding the precise mechanisms of Tregs in immune therapy is important. TILs separated from freshly excised melanoma tumours and PBMCs obtained from healthy donors were used to assess the expression levels of Tim-3 and IL-10/TGF-β of Tregs by multi-colour flow cytometry and immunohistochemistry in the presence or absence of an anti-PD-1 antibody or STAT3 inhibitor in vitro and in vivo. The mRNA level of STAT3 gene was closely associated with the expression of PD-1 and Tim-3 in melanoma patients. Tim-3 expression was significantly upregulated on Tregs after PD-1 blockade in mice treated with either induced mouse and human melanoma TILs or healthy donor PBMCs. The levels of IL-10 and TGF-β secretion in the TIL group increased compared with those in the control group, supporting a potential escape mechanism from anti-PD-1 therapy in the tumour microenvironment. Furthermore, in the presence of the STAT3 pathway inhibitor Stattic, Tim-3 and PD-1 expression on Treg was regulated on TILs from respondent mice melanoma, suggesting that STAT3 could mediate Tim-3 and PD-1 expression on Treg in tumor microenvironment. Therefore, our findings reveal the mechanism of PD-1 and Tim-3 expression in a STAT3-dependent manner on Tregs in the TME, providing further support for STAT3 as a potential transient small molecular inhibitor targets for patients suffering from melanoma. Citation Format: Lili Huang, Yu Xu, Juemin Fang, Weixing Liu, Zhuqing Liu, Qing Xu. Adaptive resistance to PD-1 blockade upregulates Tim-3 through STAT3 pathway in regulatory T cells in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1608.

Abstract 1581: Prostate tumor associated NK cells (PTANKs) acquire the decidual-like/pro-angiogenic phenotype and polarize macrophages towards the M2-like/TAM subset

Abstract Prostate cancer (PCa) remains the second most common cancer worldwide in men. Angiogenesis has been shown to play a crucial role in PCa progression in several preclinical models. Production of pro-angiogenic factors have been shown to correlate with metastasis, Gleason score and prognosis in PCa and plasma levels of VEGF were shown to be higher in patients with metastatic PCa than those with localized disease Natural killer (NK) cells, effector lymphocytes of the innate immunity, have been found to be immunosuppressed in solid cancers, including PCa. We demonstrated that NK cells in lung and colorectal cancer, acquire a pro-angiogenic phenotype, similar to NK cell within the decidua (dNK), and functionally support angiogenesis, in a TGFβ and Stat3-dependent manner. Here, we phenotypically and functionally characterized circulating NK cells from PCa patients and their interaction with endothelial cells and macrophages. NK cell subset distribution was investigated by multicolor flow cytometry (FC) for surface antigens on peripheral blood samples PCa patients. Conditioned media (CM) from FACS-sorted PTANKs were used for functional studies of angiogenesis, on human umbilical-vein endothelial cells (HUVEC), studies for macrophage recruitment (migration assay on Boyden chambers) and polarization. Molecular studies were performed by real time PCR (qPCR) on HUCECs and macrophages exposed to CM of PTANKs. Protein arrays were performed to characterize the secretome on FACS-sorted PTANKs We found that PTANKs acquire the pro-angiogenic/ decidual-likeCD56brightCD9+CD49a+CXCR4+ phenotype. The same phenotype was observed on cytolytic NK cells, from healthy donors, exposed to CMs of three different PCa cell lines. These results were confirmed also exposing heathy-donor derived NKs to CMs of 3 different prostate cancer cell lines (PC-3, DU-145, LNCaP), together with increased production of CXCL8, Angiogenin, Angiopoietin1 and reduced production of TNFα, IFNγ and GranzymeA. CMs from PTANKs support the formation on capillary-like structures on HUVEC, together with increased expression of VEGF, VEGR-2, CXCL8, ICAM-1 and VCAM-1. Secretome analysis revealed the ability of PTANKs release pro-angiogenic factors (CLXL8, MMP-1, MMP-9; uPAR) and cytokines/chemokines involved in macrophage recruitment (CCL1, CCL2, CCL5, CCL7, CCL13, CXCL1, CXCL11) and M2-like polarization (IL-10). Finally, CMs from PTANKs can recruit THP-1 monocyte and polarize THP-1 macrophage towards CD206, Arginase1, CXCL8-expressing M2-like/TAM phenotype. Our results place PTANKS as effector cells able in supporting angiogenesis in PCa by directly interaction with endothelial cells and via macrophage polarization. Citation Format: Antonino Bruno, Denisa Baci, Matteo Gallazzi, Annalisa Bosi, Angelo Naselli, Andrea Guarneri, Lorenzo Mortara, Douglas Noonan, Adriana Albini. Prostate tumor associated NK cells (PTANKs) acquire the decidual-like/pro-angiogenic phenotype and polarize macrophages towards the M2-like/TAM subset [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1581.

Testicular Lmcd1 regulates phagocytosis by Sertoli cells through modulation of NFAT1/Txlna signaling pathway.

Increased oxidative stress is well known to cause testicular dysfunction in aging males, but the detailed relationships between aging, oxidative stress, and testicular function remain to be elucidated. LIM and cysteine‐rich domains 1 (LMCD1) regulates fundamentally cellular process by interacting with transcription factors. A recent study has identified Lmcd1 as one of the most upregulated nuclear proteins associated with Sertoli cell (SC) differentiation, raising the possibility that testicular actions of LMCD1 are likely to take place. Herein, we reported that LMCD1 was exclusively expressed in the nuclei of SCs. This expression was regulated by TNF‐α signaling produced by apoptotic germ cells (GCs) and was suppressed by oxidative stress in a STAT3‐dependent manner. Ablation of endogenous LMCD1 expression caused lipid accumulation and senescence in GC co‐incubated SCs. Using a previously validated in vivo siRNA approach, we showed that LMCD1 depletion significantly impaired male fertility by inducing oligozoospermia and asthenospermia. Mechanistically, LMCD1 upregulation was associated with the nuclear enrichment of the nuclear factor of activated T cells 1 (NFAT1), a core component of Ca2+/calmodulin‐dependent pathway. LMCD1 facilitated the dephosphorylation and nuclear translocation of NFAT1, which consequently expedited the transactivation of Txlna, a binding partner of the syntaxin family essential for testicular phagocytosis, and thus promoted the removal of apoptotic GCs by phagocytic SCs. Collectively, LMCD1 may operate as a novel pretranscriptional integrator linking SC phagocytosis, lipid homeostasis, and cell senescence.

Interleukin-2 maintains the survival of interleukin-17+ gamma/delta T cells in inflammation and autoimmune diseases

There is increasing appreciation of the critical pathogenic role of IL-17 in inflammation and autoimmune diseases, which could be produced from both adaptive Th17 cells and innate γδ T cells. Existing evidences suggest that IL-2 is important for in vivo accumulation of IL-17+ γδ T cells, leaving the mechanisms still elusive. Herein, using lupus-prone MRL/lpr mice, we demonstrated that splenic γδ T cells were potent IL-17 producers at the onset of lupus, which could be diminished by in vivo IL-2 neutralization. Additional in vivo results showed that neutralization of IL-2 also significantly deleted the IL-17-producing γδ T cells in ovalbumin (OVA) /CFA-immunized B6 mice. Using splenic γδ T cells from OVA/CFA-immunized B6 mice, we further demonstrated that IL-2 could induce IL-17 production alone or together with IL-1β or IL-23 or anti-TCRγδ. Mechanism studies demonstrated that IL-2 could support the survival of γδ T cells, rather than induce the proliferation. Through specific pharmacologic inhibitor, we demonstrated that IL-2 could maintain that RORγt expression of γδ T cells in a STAT5-dependent manner. Collectively, this study suggested that the interplay between IL and 2 and other pro-inflammatory cytokines could trigger the rapid IL-17 production from innate γδ T cells, thus to orchestrate an inflammatory response before the development of adaptive Th17 cells.

Clinical and functional significance of tumor/stromal ATR expression in breast cancer patients

BackgroundMost breast cancer-associated fibroblasts (CAFs) are active and important cancer-promoting cells, with significant impact on patient prognosis. Therefore, we investigated here the role of the protein kinase ATR in breast stromal fibroblasts in the prognosis of locally advanced breast cancer patients.MethodsWe have used immunohistochemistry to assess the level of ATR in breast cancer tissues and their adjacent normal tissues. Immunoblotting as well as quantitative RT-PCR were utilized to show the role of breast cancer cells and IL-6 as well as AUF-1 in downregulating ATR in breast stromal fibroblasts. Engineered human breast tissue model was also used to show that ATR-deficient breast stromal fibroblasts enhance the growth of breast cancer cells.ResultsWe have shown that the protein kinase ATR is downregulated in cancer cells and their neighboring CAFs in breast cancer tissues as compared to their respective adjacent normal tissues. The implication of cancer cells in ATR knockdown in CAFs has been proven in vitro by showing that breast cancer cells downregulate ATR in breast fibroblasts in an IL-6/STAT3-dependent manner and via AUF-1. In another cohort of 103 tumors from locally advanced breast cancer patients, we have shown that absence or reduced ATR expression in tumoral cells and their adjacent stromal fibroblasts is correlated with poor overall survival as well as disease-free survival. Furthermore, ATR expression in CAFs was inversely correlated with tumor recurrence and progression.ConclusionATR downregulation in breast CAFs is frequent, procarcinogenic, and correlated with poor patient survival.

Interleukin-22 controls RSV production in primary human airway epithelial cells by preserving cellular autophagy

Abstract OBJECTIVE To study the ability of the host protective cytokine interleukin-22 (IL-22) to block productive infection of airway epithelial cells by Respiratory Syncytial Virus (RSV). BACKGROUND RSV infection can cause severe bronchiolitis and pneumonia in infants. However, no vaccine against RSV is currently available. RSV primarily infects both proximal and distal airways as well as alveolar epithelial cells in the lungs. IL-22 has mucosal-protective effects, known to preserve epithelial-barrier functions and promote host defense against pathogens. We, therefore, initiated studies to determine whether IL-22 can control RSV infection of airway epithelial cells (AECs). METHODS Primary human AECs differentiated under air-liquid interface (ALI) culture conditions were used for RSV infection (multiplicity of infection (MOI) 2) and then treated with IL-22 (50 ng/ml). 5-day old newborn C57BL/6 mice were intranasally infected with 1×106 PFU of RSV and then treated with an IL-22-Fc fusion protein (5 μg/mouse) intraperitoneally on day 3 p.i. Confocal imaging approach was utilized to determine co-localization of RSV and LC3B and to differentiate between autophagosomes and autolysosomes. RESULTS Treatment with IL-22 led to a significant decrease in viral load in primary human AECs as well as in vivo in mouse lungs. Confocal imaging and other approaches revealed subversion of the autophagic machinery by RSV for its own production, which was significantly inhibited by IL-22, thereby preserving autophagy in a STAT3 dependent manner. CONCLUSIONS IL-22 inhibits RSV production in both primary human airway epithelial cells and mouse lungs by preserving cellular autophagy thereby blocking hijacking of the autophagic apparatus by RSV.

IL-4 regulates resident memory CD8+ T cell formation and persistence

Abstract Tissue resident memory CD8+ T cells (TRM) persist long-term in lymphoid and non-lymphoid tissues to provide rapid, local defense against pathogens and tumors. Tissue cytokines drive the formation and persistence of CD8+ TRM. However, whether the local microenvironment can inhibit TRM formation is largely unknown. Individuals suffering from conditions associated with T helper 2 (Th2)-type immune responses, such as atopic dermatitis, asthma, and some cancers, present impaired anti-viral or anti-tumor responses. We hypothesize that Th2 cytokines inhibit the formation and/or persistence of CD8+ TRM. We demonstrate that Th2 cell supernatant downregulated TGF-β-induced CD103 expression on in vitro- and in vivo- activated CD8+ T cells in an IL-4- and Stat6-dependent manner. Moreover, IL-4 induced Eomes expression by TGF-β-stimulated CD8+ T cells. In vivo, more CD8+CD103+ TRM populated the skin of Il4ra−/− and Stat6−/− mice compared to wild-type mice at homeostasis. Additionally, CD8+ T cell activation in the presence of IL-4 impaired subsequent CD8+ TRM formation; IL-4-exposed CD8+ T cells expressed decreased CD103 in a mouse model of cutaneous inflammation, and these cells failed to persist within the skin at memory time-points. These data suggest that IL-4 counters TGF-β-mediated signals to inhibit CD8+ TRM formation/persistence and may allow identification of novel treatments to boost anti-tumor immunity and anti-viral responses in patients with type 2 inflammation.

Correction of arginine metabolism with sepiapterin—the precursor of nitric oxide synthase cofactor BH4—induces immunostimulatory-shift of breast cancer

Immunotherapy is a first-line treatment for many tumor types. However, most breast tumors are immuno-suppressive and only modestly respond to immunotherapy. We hypothesized that correcting arginine metabolism might improve the immunogenicity of breast tumors. We tested whether supplementing sepiapterin, the precursor of tetrahydrobiopterin (BH4)—the nitric oxide synthase (NOS) cofactor—redirects arginine metabolism from the pathway synthesizing polyamines to that of synthesizing nitric oxide (NO) and make breast tumors more immunogenic. We showed that sepiapterin elevated NO but lowered polyamine levels in tumor cells, as well as in tumor-associated macrophages (TAMs). This not only suppressed tumor cell proliferation, but also induced the conversion of TAMs from the immuno-suppressive M2-type to immuno-stimulatory M1-type. Furthermore, sepiapterin abrogated the expression of a checkpoint ligand, PD-L1, in tumors in a STAT3-dependent manner. This is the first study which reveals that supplementing sepiapterin normalizes arginine metabolism, improves the immunogenicity and inhibits the growth of breast tumor cells.

Unique IL-13R\u03b12/STAT3 mediated IL-13 regulation detected in lung conventional dendritic cells, 24\u2009h post viral vector vaccination

This study demonstrates that 24 h following viral vector-based vaccination IL-13Rα2 functions as a master sensor on conventional dendritic cells (cDCs), abetted by high protein stability coupled with minimal mRNA expression, to rapidly regulate DC mediated IL-13 responses at the lung mucosae, unlike IL-13Rα1. Under low IL-13, IL-13Rα2 performs as a primary signalling receptor, whilst under high IL-13, acts to sequester IL-13 to maintain homeostasis, both in a STAT3-dependent manner. Likewise, we show that viral vector-derived IL-13 levels at the vaccination site can induce differential STAT3/STAT6 paradigms in lung cDC, that can get regulated collaboratively or independently by TGF-β1 and IFN-γ. Specifically, low IL-13 responses associated with recombinant Fowlpox virus (rFPV) is regulated by early IL-13Rα2, correlated with STAT3/TGF-β1 expression. Whilst, high IL-13 responses, associated with recombinant Modified Vaccinia Ankara (rMVA) is regulated in an IL-13Rα1/STAT6 dependent manner associated with IFN-γR expression bias. Different viral vaccine vectors have previously been shown to induce unique adaptive immune outcomes. Taken together current observations suggest that IL-13Rα2-driven STAT3/STAT6 equilibrium at the cDC level may play an important role in governing the efficacy of vector-based vaccines. These new insights have high potential to be exploited to improve recombinant viral vector-based vaccine design, according to the pathogen of interest and/or therapies against IL-13 associated disease conditions.

STAT3 modulates reprogramming efficiency of human somatic cells; insights from autosomal dominant Hyper IgE syndrome caused by STAT3 mutations.

ABSTRACTHuman induced pluripotent stem cell (iPSC) technology has opened exciting opportunities for stem-cell-based therapy. However, its wide adoption is precluded by several challenges including low reprogramming efficiency and potential for malignant transformation. Better understanding of the molecular mechanisms of the changes that cells undergo during reprograming is needed to improve iPSCs generation efficiency and to increase confidence for their clinical use safety. Here, we find that dominant negative mutations in STAT3 in patients with autosomal-dominant hyper IgE (Job's) syndrome (AD-HIES) result in greatly reduced reprograming efficiency of primary skin fibroblasts derived from skin biopsies. Analysis of normal skin fibroblasts revealed upregulation and phosphorylation of endogenous signal transducer and activator of transcription 3 (STAT3) and its binding to the NANOG promoter following transduction with OKSM factors. This coincided with upregulation of NANOG and appearance of cells expressing pluripotency markers. Upregulation of NANOG and number of pluripotent cells were greatly reduced throughout the reprograming process of AD-HIES fibroblasts that was restored by over-expression of functional STAT3. NANOGP8, the human-specific NANOG retrogene that is often expressed in human cancers, was also induced during reprogramming, to very low but detectable levels, in a STAT3-dependent manner. Our study revealed the critical role of endogenous STAT3 in facilitating reprogramming of human somatic cells.

Abstract B59: IL-6 regulates CTLA4 expression on CD4+ T-cells and dual antibody blockade of IL-6 and CTLA4 leads to tumor regression in an orthotopic murine model of pancreatic ductal adenocarcinoma

Abstract Immune checkpoint blockade therapy has shown promise in many cancers. However, pancreatic ductal adenocarcinoma (PDAC) has proven refractory to most targeted and immune-based therapies. Previous research by our group and others demonstrated immune suppression in PDAC driven by activated fibroblasts, known as pancreatic stellate cells (PSCs). Specifically, our lab has demonstrated expansion of myeloid-derived suppressor cells (MDSCs) by PSCs in an interleukin-6 (IL-6) and STAT3 dependent manner (Mace T, Cancer Res 2013), while IL-6 blockade could enhance the in vivo efficacy of anti-PD-L1 antibody in multiple murine PDAC models (Mace T, Gut 2016). In addition, analysis of blood from n=73 treatment-naïve patients with metastatic PDAC revealed higher plasma IL-6, and circulating CD8+CTLA4+ T cells were associated with worse overall survival (Farren M, Clin Cancer Res 2015). Given these data, we hypothesized that blocking IL-6 in pancreatic cancer could shift the balance of effector and regulatory immune responses and enhance the efficacy of anti-CTLA4 based therapies. Human PBMCs were isolated from healthy donor blood and RossetteSep T-cell enrichment cocktail was used to enrich for CD3+ T cells. Enriched populations or bulk PBMCs were stimulated with IL-6. Cells were analyzed by flow cytometry for CTLA4 expression. In vivo studies were performed by orthotopically injecting KPC-luciferase cells into the pancreas of C57BL/6 mice. Tumor growth was monitored by bioluminescent imaging (BLI), and mice were treated with antibodies against IL-6 and CTLA4 or isotype controls. At the conclusion of in vivo studies, pancreas, liver, and spleen were harvested and analyzed using a combination of immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry. Stimulation of human PBMCs or enriched T cells with IL-6 resulted in upregulation of CTLA4 on CD4+CTLA4+ T cells and increased the percentage of CD4 cells positive for CTLA4. We observed no change associated with CD8+ T cells. Dual antibody blockade of IL-6 and CTLA4 in an orthotopic murine model of PDAC led to significant tumor regression as determined by BLI in comparison to isotype treatment, with significantly lower end study tumor weights. Addition of CXCR3 blocking antibodies to mice receiving the combination therapy abrogated this antitumor response. IHC analysis of tumors showed combination-treated tumors to have higher infiltration of CD8+ cells in comparison to isotype or single-agent treatment. We demonstrate, for the first time, the ability of IL-6 to regulate CTLA4 expression on CD4+ T cells, unveiling a new dynamic in PDAC-associated immune suppression. Our in vivo data demonstrate the ability of dual IL-6 and CTLA4 blockade to lead to PDAC tumor regression. These data, in combination with our previous research, strongly suggest that blocking IL-6 in the clinical setting will enhance the antitumor effect of anti-CTLA4 antibodies and other immune-based therapies in PDAC. Citation Format: Michael B. Ware, Christopher McQuinn, Thomas A. Mace, Jacob Bowers, Reena Shakya, Brad Farris, Gregory Young, William E. Carson III, Chrystal M. Paulos, Bassel El-Rayes, Gregory B. Lesinski. IL-6 regulates CTLA4 expression on CD4+ T-cells and dual antibody blockade of IL-6 and CTLA4 leads to tumor regression in an orthotopic murine model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B59.

MKL1 promotes endothelial-to-mesenchymal transition and liver fibrosis by activating TWIST1 transcription

Excessive fibrogenic response in the liver disrupts normal hepatic anatomy and function heralding such end-stage liver diseases as hepatocellular carcinoma and cirrhosis. Sinusoidal endothelial cells contribute to myofibroblast activation and liver fibrosis by undergoing endothelial-mesenchymal transition (EndMT). The underlying mechanism remains poorly defined. Here we report that inhibition or endothelial-specific deletion of MKL1, a transcriptional modulator, attenuated liver fibrosis in mice. MKL1 inhibition or deletion suppressed EndMT induced by TGF-β. Mechanistically, MKL1 was recruited to the promoter region of TWIST1, a master regulator of EndMT, and activated TWIST1 transcription in a STAT3-dependent manner. A small-molecule STAT3 inhibitor (C188-9) alleviated EndMT in cultured cells and bile duct ligation (BDL) induced liver fibrosis in mice. Finally, direct inhibition of TWIST1 by a small-molecule compound harmine was paralleled by blockade of EndMT in cultured cells and liver fibrosis in mice. In conclusion, our data unveil a novel mechanism underlying EndMT and liver fibrosis and highlight the possibility of targeting the STAT3-MKL1-TWIST1 axis in the intervention of aberrant liver fibrogenesis.

CDK5RAP3 is a co-factor for the oncogenic transcription factor STAT3

The transcription factor STAT3 regulates genes governing critical cellular processes such as proliferation, survival, and self-renewal. While STAT3 transcriptional function is activated rapidly and transiently in response to physiologic signals, through a variety of mechanisms it can become constitutively activated in the pathogenesis of cancer. This leads to chronic expression of genes that underlie malignant cellular behavior. However, STAT3 is known to interact with other proteins, which may modulate its function. Understanding these interactions can provide insights into novel aspects of STAT3 function and may also suggest strategies to therapeutically target the large number of cancers driven by constitutively activated STAT3. To identify critical modulators of STAT3 transcriptional function, we performed an RNA-interference based screen in a cell-based system that allows quantitative measurement of STAT3 activity. From this approach, we identified CDK5 kinase regulatory-subunit associated protein 3 (CDK5RAP3) as an enhancer of STAT3-dependent gene expression. We found that STAT3 transcriptional function is modulated by CDK5RAP3 in cancer cells, and silencing CDK5RAP3 reduces STAT3-mediated tumorigenic phenotypes including clonogenesis and migration. Mechanistically, CDK5RAP3 binds to STAT3-regulated genomic loci, in a STAT3-dependent manner. In primary human breast cancers, the expression of CDK5RAP3 expression was associated with STAT3 gene expression signatures as well as the expression of individual STAT3 target genes. These findings reveal a novel aspect of STAT3 transcriptional function and potentially provide both a biomarker of enhanced STAT3-dependent gene expression as well as a unique mechanism to therapeutically target STAT3.

STAT3 Inhibition Partly Abolishes IL-33-Induced Bone Marrow-Derived Monocyte Phenotypic Transition into Fibroblast Precursor and Alleviates Experimental Renal Interstitial Fibrosis.

Previous studies of Jak-STAT inhibitors have shown promise in treating kidney diseases. The activation of Jak-STAT components is important in cell fate determination in many cell types, including bone marrow-derived cells, which are important contributors in renal interstitial fibrosis. In this study, we tested the effect of a new STAT3 inhibitor, BP-1-102, on monocyte-to-fibrocyte transition and the progression of renal interstitial fibrosis. We tested the effect of BP-1-102 in a mouse model of unilateral ureteral obstruction in vivo and IL-33-treated bone marrow-derived monocytes in vitro. BP-1-102 treatment alleviated renal interstitial fibrosis, reduced collagen deposition and extracellular matrix protein production, inhibited inflammatory cell infiltration, suppressed the percentage of CD45+ PDGFRβ+, CD45+ CD34- Col I+ and CD45+ CD11b+ Col I+ cells within the obstructed kidney and reduced the mRNA levels of the proinflammatory and profibrotic cytokines IL-1β, TGF-β, TNF-α, ICAM-1, and CXCL16. In vitro, BP-1-102 inhibited the IL-33-induced phenotypic transition into fibroblast precursors in bone marrow-derived monocytes, marked by reduced CD45+ CD34- Col I+ and CD45+ CD11b+ Col I+ cell percentage. Our results indicate a potential mechanism by which the STAT3 inhibitor BP-1-102 inhibits bone marrow-derived monocyte transition into fibroblast precursors in an IL-33/STAT3-dependent manner and thereby alleviates renal interstitial fibrosis.

A Sexually Dimorphic Role for STAT3 in Sonic Hedgehog Medulloblastoma.

Medulloblastoma is the most common malignant brain tumor in children and represents 20% of all pediatric central nervous system neoplasms. While advances in surgery, radiation and chemotherapy have improved overall survival, the lifelong sequelae of these treatments represent a major health care burden and have led to ongoing efforts to find effective targeted treatments. There is a well-recognized male bias in medulloblastoma diagnosis, although the mechanism remains unknown. Herein, we identify a sex-specific role for the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) in the Sonic Hedgehog (SHH) medulloblastoma subgroup. Specific deletion of Stat3 from granule cell precursors in a spontaneous mouse model of SHH medulloblastoma completely protects male, but not female mice from tumor initiation. Segregation of SHH medulloblastoma patients into high and low STAT3 expressing cohorts shows that low STAT3 expression correlates with improved overall survival in male patients. We observe sex specific changes in IL-10 and IL-6 expression and show that IL-6 stimulation enhances SHH-mediated gene transcription in a STAT3-dependent manner. Together these data identify STAT3 as a key molecule underpinning the sexual dimorphism in medulloblastoma.

A crucial role of fibroblast growth factor 2 in the differentiation of hair follicle stem cells toward endothelial cells in a STAT5-dependent manner

Fibroblast growth factor (FGF2) is reported to affect the proliferation, differentiation, and survival abilities of stem cells. In this study, we hypothesize that FGF2 might promote the differentiation of hair follicle stem cell (HFSCs) into endothelial cells (ECs), in a manner dependent on STAT5 activation. We first treated human HFSCs with recombinant human FGF2 to determine the involvement of FGF2 in the differentiation of HFSCs. Then the expression of EC-specific markers including von Willebrand factor (vWF), VE-cadherin, CD31, FLT-1, KDR and Tie2 was evaluated using immunofluorescence and flow cytometry, while the expression of HFSC-specific markers such as K15, K19, Lgr5, Sox9 and Lhx2 was determined by flow cytometry. Next, in vitro tube formation was performed to confirm the function of FGF2, and low-density lipoprotein (LDL) uptake by ECs and HFSCs was studied by Dil-acetylated LDL assay. In addition, we transduced FGF2-treated HFSCs with constitutive-active or dominant-negative STAT5A adenovirus vectors. FGF2 up-regulated the expression of EC-specific markers, and promoted the differentiation of HFSCs into ECs, tube formation and LDL uptake. The phosphorylated STAT5 was translocated into the nucleus of HFSCs after FGF2 treatment, but this translocation was blocked by the dominant-negative STAT5A mutant. FGF2 increased the differentiation potential through the activation of STAT5 in vivo. Taken together, we find that FGF2 promotes the differentiation of HFSCs into ECs via activated STAT5, which gives a new perspective on the role of FGF2 in the development of ischemic vascular disease.

AMPK Alpha-1 Intrinsically Regulates the Function and Differentiation of Tumor Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSC) represent a primary mechanism of immune evasion in tumors and have emerged as a major obstacle for cancer immunotherapy. The immunoinhibitory activity of MDSC is tightly regulated by the tumor microenvironment and occurs through mechanistic mediators that remain unclear. Here, we elucidated the intrinsic interaction between the expression of AMP-activated protein kinase alpha (AMPKα) and the immunoregulatory activity of MDSC in tumors. AMPKα signaling was increased in tumor-MDSC from tumor-bearing mice and patients with ovarian cancer. Transcription of the Ampkα1-coding gene, Prkaa1, in tumor-MDSC was induced by cancer cell-derived granulocyte-monocyte colony-stimulating factor (GM-CSF) and occurred in a Stat5-dependent manner. Conditional deletion of Prkaa1 in myeloid cells, or therapeutic inhibition of Ampkα in tumor-bearing mice, delayed tumor growth, inhibited the immunosuppressive potential of MDSC, triggered antitumor CD8+ T-cell immunity, and boosted the efficacy of T-cell immunotherapy. Complementarily, therapeutic stimulation of AMPKα signaling intrinsically promoted MDSC immunoregulatory activity. In addition, Prkaa1 deletion antagonized the differentiation of monocytic-MDSC (M-MDSC) to macrophages and re-routed M-MDSC, but not granulocytic-MDSC (PMN-MDSC), into cells that elicited direct antitumor cytotoxic effects through nitric oxide synthase 2-mediated actions. Thus, our results demonstrate the primary role of AMPKα1 in the immunosuppressive effects induced by tumor-MDSC and support the therapeutic use of AMPK inhibitors to overcome MDSC-induced T-cell dysfunction in cancer. SIGNIFICANCE: AMPKα1 regulates the immunosuppressive activity and differentiation of tumor-MDSC, suggesting AMPK inhibition as a potential therapeutic strategy to restore protective myelopoiesis in cancer.

Ozone Inhalation Attenuated the Effects of Budesonide on Aspergillus fumigatus-Induced Airway Inflammation and Hyperreactivity in Mice.

Inhaled glucocorticoids form the mainstay of asthma treatment because of their anti-inflammatory effects in the lung. Exposure to the air pollutant ozone (O3) exacerbates chronic airways disease. We and others showed that presence of the epithelial-derived surfactant protein-D (SP-D) is important in immunoprotection against inflammatory changes including those induced by O3 inhalation in the airways. SP-D synthesis requires glucocorticoids. We hypothesized here that O3 exposure impairs glucocorticoid responsiveness (including SP-D production) in allergic airway inflammation. The effects of O3 inhalation and glucocorticoid treatment were studied in a mouse model of allergic asthma induced by sensitization and challenge with Aspergillus fumigatus (Af) in vivo. The role of O3 and glucocorticoids in regulation of SP-D expression was investigated in A549 and primary human type II alveolar epithelial cells in vitro. Budesonide inhibited airway hyperreactivity, eosinophil counts in the lung and bronchoalveolar lavage (BAL) and CCL11, IL-13, and IL-23p19 release in the BAL of mice sensitized and challenged with Af (p < 0.05). The inhibitory effects of budesonide were attenuated on inflammatory changes and were completely abolished on airway hyperreactivity after O3 exposure of mice sensitized and challenged with Af. O3 stimulated release of pro-neutrophilic mediators including CCL20 and IL-6 into the airways and impaired the inhibitory effects of budesonide on CCL11, IL-13 and IL-23. O3 also prevented budesonide-induced release of the immunoprotective lung collectin SP-D into the airways of allergen-challenged mice. O3 had a bi-phasic direct effect with early (<12 h) inhibition and late (>48 h) activation of SP-D mRNA (sftpd) in vitro. Dexamethasone and budesonide induced sftpd transcription and translation in human type II alveolar epithelial cells in a glucocorticoid receptor and STAT3 (an IL-6 responsive transcription factor) dependent manner. Our study indicates that O3 exposure counteracts the effects of budesonide on airway inflammation, airway hyperreactivity, and SP-D production. We speculate that impairment of SP-D expression may contribute to the acute O3-induced airway inflammation. Asthmatics exposed to high ambient O3 levels may become less responsive to glucocorticoid treatment during acute exacerbations.

The protective role of NR4A3 in acute myocardial infarction by suppressing inflammatory responses via JAK2-STAT3/NF-κB pathway

Inflammatory responses play a critical role in left ventricular remodeling after acute myocardial infarction (AMI). NR4A3, a member of the NR4A orphan nucleus receptor family, has recently emerged as a therapeutic target for treatment of inflammation. This aim of this study is to explore the therapeutic effect of NR4A3 in cardiac remodeling post AMI. Male C57BL/6 mice were administered with lentiviral over-expression of NR4A3 (lenti-NR4A3) or empty vector (lenti-con) 7 days before coronary artery ligation. H9c2 cardiomyocytes deprived of serum were used to mimic ischemic conditions in vivo. Lenti-NR4A3 treatment significantly repressed neutrophil infiltration in the myocardium, reduced infarct size, and attenuated the reduction of left ventricular function after AMI. Furthermore, NR4A3 over-expression inhibited the NF-κB (IκB) signaling by decreasing IκBα phosphorylation and by inhibiting the translocation of p65 to the nucleus. Meanwhile, NR4A3 over-expression also increases the activity of JAK2-STAT3 signaling in mouse hearts after AMI. The inhibitory effect of NR4A3 on NF-κB activation was almost completely abolished by the JAK2 inhibitor AG490, indicating that NR4A3 prevented serum deprivation induced NF-κB activation in a STAT3 dependent manner. These findings provide novel evidence that NR4A3 could inhibit post-AMI inflammation responses via JAK2-STAT3/NF-κB signaling and may well be a therapeutic target for cardiac remodeling after AMI.