Expression of STAT1 and STAT2 in malignant melanoma does not correlate with response to interferon-alpha adjuvant therapy

Interferon-alpha (IFN-) is used as an adjuvant therapy in patients with malignant melanoma and who have undergone surgical resection of high-risk lesions. Defective expression or activation of STAT1 or STAT2 has been shown to correlate with IFN- or resistance in vitro; however, recent data from our laboratory suggest that the anti-tumor effects of IFN- are dependent on STAT1 signaling within host immune cells. We measured STAT1 and STAT2 expression in 28 melanoma biopsies (8 cutaneous lesions; 1 lung metastasis; 19 nodal metastases) obtained from patients prior to the initiation of adjuvant IFN- therapy. Disease recurrence following IFN- treatment did not correlate with the staining intensity of either STAT1 (P=0.61) or STAT2 (P=0.52). Tumors with minimal STAT1 or STAT2 expression (<20% positive) were present in four patients with tumor-positive lymph nodes, who exhibited prolonged relapse-free survival (>44 months) following adjuvant therapy. Conversely, high levels of STAT1 were present in a patient who recurred during the course of IFN- therapy. A case study of one patient who experienced recurrent disease during IFN- treatment revealed that STAT1 levels were greater in the recurrent tumor when compared to the original lesion. These studies provide direct evidence to suggest that levels of STAT1 and STAT2 within the tumor do not influence a patients response to adjuvant IFN-.     

Gap junctions mediate STAT5-independent β-casein expression in CID-9 mammary epithelial cells

Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent β-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed β-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed β-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced β-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed β-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.     

Rap1A regulates generation of T regulatory cells via LFA-1-dependent and LFA-1-independent mechanisms

The small GTPase Rap1A has a critical role in regulating cell-matrix and cell-cell adhesion. In T lymphocytes, Rap1A mediates LFA-1 activation and LFA-1-mediated adhesion. LFA-1 reduces the threshold of TCR signals for low affinity ligands. Previously, we determined that mice expressing constitutively active Rap1A on T cells have increased frequency of CD103⁺ T regulatory cells (Treg). We hypothesized that Rap1A-GTP might affect the differentiation of Treg by regulating LFA-1 activation. Using Foxp3-GFP-KI, LFA-1-KO and Rap1A-GTP-Tg mice we determined that Rap1A has an active role in the development of thymic Treg but LFA-1 is not mandatory for this function. Rap1A is also involved in the generation of peripheral Treg and this effect is mediated via LFA-1-dependent and LFA-1-independent mechanisms. Identification of the signaling pathways via which Rap1-GTP contributes to the differentiation of Treg will provide new insights to the function of Rap1A and to designing targeted approaches for generation of Treg for therapeutic applications.     

Tannic acid inhibits EGFR/STAT1/3 and enhances p38/STAT1 signalling axis in breast cancer cells

Tannic acid (TA), a naturally occurring polyphenol, is a potent anti‐oxidant with anti‐proliferative effects on multiple cancers. However, its ability to modulate gene‐specific expression of tumour suppressor genes and oncogenes has not been assessed. This work investigates the mechanism of TA to regulate canonical and non‐canonical STAT pathways to impose the gene‐specific induction of G1‐arrest and apoptosis. Regardless of the p53 status and membrane receptors, TA induced G1‐arrest and apoptosis in breast cancer cells. Tannic acid distinctly modulated both canonical and non‐canonical STAT pathways, each with a specific role in TA‐induced anti‐cancer effects. Tannic acid enhanced STAT1 ser727 phosphorylation via upstream serine kinase p38. This STAT1 ser727 phosphorylation enhanced the DNA‐binding activity of STAT1 and in turn enhanced expression of p21^Waf1/Cip1. However, TA binds to EGF‐R and inhibits the tyrosine phosphorylation of both STAT1 and STAT3. This inhibition leads to the inhibition of STAT3/BCL‐2 DNA‐binding activity. As a result, the expression and mitochondrial localization of BCl‐2 are declined. This altered expression and localization of mitochondrial anti‐pore factors resulted in the release of cytochrome c and the activation of intrinsic apoptosis cascade involving caspases. Taken together, our results suggest that TA modulates EGF‐R/Jak2/STAT1/3 and P38/STAT1/p21^Waf1/Cip1 pathways and induce G1‐arrest and intrinsic apoptosis in breast carcinomas.     

Sodium butyrate inhibits interferon-gamma induced indoleamine 2,3-dioxygenase expression via STAT1 in nasopharyngeal carcinoma cells

Aims

Indoleamine 2,3-dioxygenase (IDO) inhibits T-cell proliferation by catalyzing the conversion of l-tryptophan to l-kynurenine. IDO-induced immune tolerance weakens the clinical outcomes of immunotherapies. Sodium butyrate (NaB), one of the histone deacetylase inhibitors (HDACIs), has potential anti-tumor effects. Our previous studies revealed that NaB could inhibit IFN-γ induced IDO expression in nasopharyngeal carcinoma cells, CNE2. In the present study, we aim to investigate to the mechanism of NaB interfering with the interferon-gamma (IFN-γ)-mediated IDO expression signaling transduction.

Main methods

IDO expression and STAT1 phosphorylation in CNE2 cells were analyzed by western blotting and STAT1 acetylation was evaluated by immunoprecipitation. STAT1 nuclear translocation and NF-κB activity were detected by transient transfection and reporter gene assay.

Key findings

We found that NaB inhibited IFN-γ-induced IDO expression in CNE2 cells via decreasing phosphorylation and nuclear translocation of STAT1, but not via down-regulation of IFN-γ-receptor (IFNGR). Immunoprecipitation assays revealed that NaB increased STAT1 acetylation. Furthermore, NaB elevated the activity of NF-κB in CNE2 cells, and blocking the NF-κB activity had no effect on the IFN-γ-induced IDO expression.

Significance

These results suggest that NaB inhibited IFN-γ-induced IDO expression via STAT1 increased acetylation, decreased phosphorylation, and reduced nuclear translocation. These provided new evidence for the anti-tumor action of NaB and potential drug targets to reduce the IDO-induced immune tolerance.     

Evidence for interleukin-1-independent stimulation of interleukin-12 and down-regulation by interleukin-10 in Helicobacter pylori-infected murine dendritic cells deficient in the interleukin-1 receptor

Helicobacter pylori infection is characterized by infiltration of cells of the immune system, including dendritic cells, into the gastric mucosa. During chronic inflammation with Helicobacter pylori infection, a variety of cytokines are secreted into the mucosa, including interleukin-1beta (IL-1beta). The role of IL-1 in H. pylori infection was investigated using bone-marrow-derived dendritic cells from wild-type and IL-1 receptor-deficient (IL-1R-/-) mice. Dendritic cells were incubated with H. pylori at a multiplicity of infection of 10 and 100, and cytokine production evaluated. Helicobacter pylori SS1, H. pylori SD4, and an isogenic cagE mutant of SD4 stimulated IL-12, IL-6, IL-1beta, IL-10, and tumor necrosis factor-alpha at comparable levels in dendritic cells from both wild-type and IL-1R-/- mice. IL-10 production required the higher inoculum, while IL-12 was decreased at this bacterial load. Pretreatment of dendritic cells with an antibody to IL-10 resulted in an increased production of IL-12, confirming the down-regulation of IL-12 by IL-10. cagE was required for maximum stimulation of IL-12 by H. pylori. We speculate that the down-regulation of IL-12 by IL-10 at the higher multiplicity of infection represents the modulation of the host inflammatory response in vivo by H. pylori when the bacterial load is high, allowing for persistent colonization of the gastric mucosa.     

Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer

The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway. Although several studies have indicated that IFN-alpha and IFN-beta can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN-alpha or IFN-beta led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2: STAT6 complexes, which led to the secretion of IL-1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra. The present study suggests a novel function of IFN-alpha and IFN-beta signalling in human hepatocytes. Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.     

Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications for tumor immunotherapy

Programmed death receptor ligand 1 (PD-L1, also called B7-H1) is a recently described B7 family member. In contrast to B7-1 and B7-2, PD-L1 does not interact with either CD28 or CTLA-4. To date, one specific receptor has been identified that can be ligated by PD-L1. This receptor, programmed death receptor 1 (PD-1), has been shown to negatively regulate T-cell receptor (TCR) signaling. Upon ligating its receptor, PD-L1 has been reported to decrease TCR-mediated proliferation and cytokine production. PD-1 gene–deficient mice developed autoimmune diseases, which early led to the hypothesis of PD-L1 regulating peripheral tolerance. In contrast to normal tissues, which show minimal surface expression of PD-L1 protein, PD-L1 expression was found to be abundant on many murine and human cancers and could be further up-regulated upon IFN- stimulation. Thus, PD-L1 might play an important role in tumor immune evasion. This review discusses the currently available data concerning negative T-cell regulation via PD-1, the blockade of PD-L1/PD-1 interactions, and the implications for adoptive T-cell therapies.     

Modulation of TNF-alpha gene expression by IFN-gamma and pamidronate in murine macrophages: regulation by STAT1-dependent pathways

Aminobisphosphonates are drugs used in the treatment of hypercalcemia, Paget's disease, osteoporosis, and malignancy. Some patients treated with aminobisphosphonates have a transient febrile reaction that may be caused by an increased serum concentration of proinflammatory cytokines. Aminobisphosphonates induce the production of certain proinflammatory cytokines in vitro, especially in cells of monocytic lineage. A unique feature of aminobisphosphonates is that they bind the Vgamma2Vdelta2 class of T cells, which are found only in primates, and stimulate cytokine production. The effects of aminobisphosphonates on other cells, including macrophages, are incompletely understood. We show in this study that treatment of murine macrophages with pamidronate, a second generation aminobisphosphonate, induces TNF-alpha production. Furthermore, pretreatment of murine macrophages with pamidronate before stimulation with IFN-gamma significantly augments IFN-gamma-dependent production of TNF-alpha. This pamidronate-mediated augmentation of TNF-alpha production results in sustained phosphorylation of the tyrosine residue at position 701 of STAT1 after IFN-gamma treatment. Our data suggest that this sustained phosphorylation results from inhibition of protein tyrosine phosphatase activity. We also show that pamidronate treatment increases TNF-alpha production in vivo in mice. Pamidronate-augmented TNF-alpha production by macrophages might be a useful strategy for cytokine-based anticancer therapy.     

STAT1 is a key gene in a gene regulatory network related to immune phenotypes in bladder cancer: An integrative analysis of multi-omics data

The immunophenotype of bladder cancer plays a pivotal role in the prognosis of cancer, but the effect of different epigenetic factors on different immunophenotypes in bladder tumours remains unclear. This study used multi-omics data analysis to provide molecular basis support for different immune phenotypes. Unsupervised cluster analysis revealed distinct subclusters with higher (subcluster B2) or lower cytotoxic immune phenotypes (subcluster A1) related to PD-L1 and IFNG expression. Mutational landscape analyses showed that the mutation level of TP53 in subcluster B1 was highest than other subclusters, and subcluster B1 had a lower frequency of concurrent mutation than subcluster A2. A total of 2364 differentially expressed genes were identified between subclusters A2 and B1, and the main functions of the up-regulated genes in subcluster B1 were enriched in the activation of T cells and other related pathways. We found that STAT1 was a key gene in a gene regulatory network related to immune phenotypes in bladder cancer. Finally, we constructed a prognostic prediction model by LASSO Cox regression which could distinguish high-risk and low-risk cases significantly. In conclusion, the present study addressed a field synopsis between genetic and epigenetic events in immune phenotypes of bladder cancer.