Phosphorylation Of STAT Proteins Research Articles

LncRNA ALMS1-IT1 modulates ferroptosis and immune evasion in colorectal cancer through activating STAT3.

Colorectal cancer (CRC) represents a significant malignancy within the digestive system, characterized by high incidence and mortality rates. In recent years, molecular targeted therapy has been introduced as a supplementary strategy in CRC management, complementing traditional modalities such as surgery, radiation and chemotherapy. The identification of novel therapeutic targets for CRC remains critically important. Ferroptosis, a unique form of programmed cell death distinct from apoptosis and necrosis, is characterized by cellular damage resulting from iron-induced lipid peroxidation, leading to cell death. This study utilizes a combination of bioinformatics analysis and clinical specimen validation to demonstrate that the long non-coding RNA (lncRNA) ALMS1-IT1 is significantly upregulated in CRC tissues and strongly associated with ferroptosis. Through a series of experimental investigations, we have determined that ALMS1-IT1 negatively regulates ferroptosis in CRC cells, thereby promoting cancer growth and metastasis, acting as an oncogenic factor. Furthermore, we explored the molecular interactions of ALMS1-IT1, revealing its role in activating STAT3 protein phosphorylation. This activation enhances the immune evasion capabilities of CRC cells. Rescue experiments indicated that STAT3 activation is essential for ALMS1-IT1's suppression of ferroptosis, immune evasion and oncogenic behaviour in CRC. Our findings underscore the critical biological role of ALMS1-IT1 in the progression of CRC and suggest its potential as a target for drug development.

RuBi-Ruxolitinib: A Photoreleasable Antitumor JAK Inhibitor.

We describe the synthesis and biological testing of ruthenium-bipyridine ruxolitinib (RuBiRuxo), a photoreleasable form of ruxolitinib, a JAK inhibitor used as an antitumoral agent in cutaneous T-cell lymphomas (CTCL). This novel caged compound is synthesized efficiently, is stable in aqueous solution at room temperature, and is photoreleased rapidly by visible light. Irradiation of RuBiRuxo reduces cell proliferation and induces apoptosis in a light- and time-dependent manner in a CTCL cell line. This effect is specific and is mediated by a decreased phosphorylation of STAT proteins. Our results demonstrate the potential of ruthenium-based photocompounds and light-based therapeutic approaches for the potential treatment of cutaneous lymphomas and other pathologies.

Chemerin promotes invasion of oral squamous cell carcinoma by stimulating IL-6 and TNF-α production via STAT3 activation.

Elevated levels of adipokine chemerin have been identified in oral squamous cell carcinoma (OSCC) and found to be associated with metastasis to the cervical lymph nodes. The underlying mechanism through which chemerin affects OSCC progression is unclear. The aims of this study were firstly to determine chemerin levels and cytokine concentrations in serum from patients with OSCC and in OSCC cell cultures, and secondly to observe chemerin effects on OSCC cell cytokine secretion, migration, and invasion in vitro. Serum samples were collected from 20 patients diagnosed with OSCC, including groups with (LN+) and without (LN-) cervical lymph node metastasis. A Luminex liquid suspension assay was used to quantify serum concentrations of 27 types of cytokines. Correlations between chemerin and cytokines (i.e., IL-6, IL-15, GM-CSF, RANTES, TNF-α, and VEGF) were analyzed. ELISAs (enzyme-linked immunosorbent assays) were used to determine concentrations of chemerin and selected cytokines in serum and in supernatants of OSCC cell cultures (SCC9 and SCC25 cell lines). OSCC cells were stimulated with human recombinant chemerin, STAT3 inhibitor, or IL-6 together with TNF-α neutralizing antibodies. Phosphorylated STAT3 protein levels were measured with western blot analysis. OSCC cell migration and invasion were investigated with Transwell assays. Compared to the LN- group, OSCC patients with cervical lymph node metastasis had higher levels of IL-6 (P = 0.006), IL-15 (P = 0.020), GM-CSF (P = 0.036), RANTES (P = 0.032), TNF-α (P = 0.005), VEGF (P = 0.006), and chemerin (P = 0.001). Patients' serum chemerin levels correlated directly with IL-6, GM-CSF, TNF-α, and VEGF levels in OSCC patients. Exogenous recombinant chemerin treatment promoted secretion of IL-6 and TNF-α via activation of STAT3 in OSCC cells. Chemerin induced OSCC-cell migration and invasion, and these effects were reduced by IL-6 and TNF-α neutralizing antibodies. Our findings indicate that chemerin may play a role in advancing OSCC progression by increasing production of IL-6 and TNF-α, perhaps via a mechanism involving STAT3 signaling.

Abstract 7225: Combination JAK1/2 and CDK8/19 inhibition demonstrates enhanced efficacy in myeloproliferative neoplasms

Abstract Myeloproliferative neoplasms (MPNs) are characterized by aberrant activation of the JAK-STAT pathway. Ruxolitinib (RUX) is a JAK1/2 inhibitor used to treat myelofibrosis (MF) and hydroxyurea-resistant/intolerant polycythemia vera. While RUX has demonstrated important clinical benefits, a large proportion of patients have persistent disease manifestations despite therapy. CDK8 regulates phosphorylation of STAT proteins in a RUX-independent manner; thus, CDK8 inhibition may alter downstream STAT target gene expression. RVU120 is a highly selective and potent CDK8/19 inhibitor that inhibits STAT1/5 phosphorylation. We hypothesized that the combination of RUX and RVU120 would act in a cooperative manner to attenuate JAK-STAT signaling and reduce MPN phenotypes in vivo. Using JAK2V617F mutant leukemic cell lines, we identified dose-responsive changes in viability with RUX and RVU120 in all cell lines. Pharmacodynamic analysis validated that STAT5 protein phosphorylation was decreased by RUX/RVU120, although at different phosphorylation sites by RUX (pTYR694) and RVU120 (pSER726/731); exposure to RUX+RVU120 inhibited phosphorylation at both sites. Notably, analysis of combinatorial effects of RUX+RVU120 on viability demonstrated drug synergies (combinatorial index <1) in RUX-naïve and resistant SET-2 cells. Nascent RNA expression data exploring JAK-STAT and mediator-related pathways of pathogenesis will be presented at the meeting. For in vivo studies, we used two previously-described murine models of MF and PV. Briefly, retrovirally-transfected (MSCV-MPLW515L-IRES-GFP) or Jak2V617F-conditional knock-in bone marrow (BM) was intravenously injected into lethally-irradiated congenic recipients. Mice were randomized and treated for several weeks with vehicle (VEH), single-agent RUX/RVU120, or RUX+RVU120 arms. Spleen weights between VEH and RUX/RVU120 treated mice at time of terminal sacrifice were significantly reduced across both models (p⇐0.009). In MPLW515L mice, WBCs and PB GFP% were significantly reduced (p<0.002) and histopathology demonstrated near-elimination of BM reticulin fibrosis and improved trilineage hematopoiesis between RUX+RVU120 and RUX arms. Data on overall survival, cytokine profiles, and the impact of RVU120 on dynamic gene expression will be presented at the meeting. The combination of RVU120 and RUX demonstrated biochemical synergy and differential inhibition of STAT5 phosphorylation in vitro. Further, in vivo treatment with RVU120/RUX+RVU120 resulted in significant reductions of disease manifestation (splenomegaly, WBC, fibrosis scoring, hematopoiesis) when compared to VEH/RUX. These data nominate JAK1/2 and CDK8/19 inhibition as a potential novel therapeutic strategy in MPNs; further work on nascent RNA expression and cytokine profiles will potentially elucidate additional mechanisms of action. Citation Format: Zachary Zaroogian, Elżbieta Adamczyk, Adrianna Moszyńska, Marta Obacz, Urszula Pakulska, Benjamin Durham, Milena Mazan, Shoron Mowla, Katarzyna Wnęk, Amritha Varshini Hanasoge Somasundara, Beata Barczuk, Aniela Gołas, Ross Levine, Tomasz Rzymski, Raajit Rampal. Combination JAK1/2 and CDK8/19 inhibition demonstrates enhanced efficacy in myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7225.

Abstract 665: MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib

Abstract Myelofibrosis (MF) is a pathological chronic bone marrow (BM) condition characterized by abnormal growth of erythroid and megakaryocytic precursors and deposition of fibrotic tissue. The JAK/STAT pathway plays an important role in MF (Cardoso 2015), and multiple JAK1/2 inhibitors (JAKis) are approved for MF treatment. Despite initially limiting disease symptoms, JAKis often fail due to persistent JAK2 activation and survival of MF-driving cells, hence new treatment strategies are highly needed. PIM kinases also contribute to MF pathogenesis. PIM1 is highly overexpressed in granulocytes, peripheral blood mononuclear cells (PBMCs), and BM of patients with MF. PIM1/2 expression is dependent upon JAK2 signaling in myeloproliferative neoplasms (MPNs). Hence, PIM inhibitors represent potential therapeutic targets for MPNs and have been evaluated in several preclinical and clinical studies (Rampal 2021). MEN1703/SEL24 (MEN) is an oral, first-in-class, dual PIM/FLT3 kinase inhibitor in development for hematologic malignancies. This study aims to investigate the efficacy of MEN alone and in combination with the JAKi ruxolitinib (RUX) in preclinical MF models and to elucidate the underlying signaling pathways. MF cell lines (JAK2V617F and JAK2 wild type) were used in MTS assays to assess the in vitro cytotoxicity of MEN alone and in combination with RUX, compared with the PIM inhibitor TP-3654 (Dutta 2022). Cell cycle distribution and signaling pathways were analyzed via propidium iodide/FACS analysis and immunocapillary electrophoresis, respectively, upon treatment with the single agents and MEN + RUX. In vivo efficacy was assessed in a murine xenograft model treated with MEN +/- RUX. In vitro studies showed single agent MEN activity with an IC50 ranging from 0.3 to 2.3 µM. Moreover, a synergistic drug interaction was found with MEN + RUX in all tested cell lines (Combination Index [CI] range 0.41-0.80). MEN presented cytotoxic and cytostatic capacity, inducing apoptosis and arresting a proportion of cells in G2/M phase. This effect was maintained in combination with RUX. Signaling pathway analysis showed that MEN inhibits phosphorylation of STAT proteins and decreases Mcl1 protein expression. These effects were enhanced with MEN + RUX. Compared with TP-3654, MEN was more effective both as a single agent (IC50 2-3 fold lower than TP-3654) and in combination with RUX (mean CI of 0.77-0.80 compared to 0.79-0.98 with TP-3654). MEN showed anti-tumoral efficacy in MF preclinical models, moderately higher than TP-3654, exhibiting in vitro activity at clinically relevant concentrations. Importantly, MEN combined with the standard of care RUX was synergistic, and molecular analyses confirmed the role of PIM downstream target inhibition. Our results support the therapeutic potential and relevance of MEN in MF treatment strategies. Citation Format: Maria Luisa Iannitto, Diego Bisignano, Sonia Zicari, Francesco Belli, Alessio Fiascarelli, Simone Talucci, Giuseppe Merlino, Vittoria Nicolis di Robilant, Clelia Irrissuto, Mario Bigioni, Alessandro Bressan, Daniela Bellarosa, Monica Binaschi. MEN1703/SEL24 exhibits promising antitumoral activity in preclinical models of myelofibrosis both as single agent and combined with ruxolitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 665.

An emerging role of guanidine acetic acid in rescuing immune function injured by Aeromonas hydrophila in grass carp (Ctenopharyngodon idella)

Guanidine acetic acid (GAA) is a safe feed additive and the only precursor for the creatine synthesis of vertebrates. Aeromonas hydrophila is a major fish pathogen causing severe morbidity and mortality in the aquaculture sector. Under the current intensive farming system, A feed additive that can improve fish disease resistance is urgently needed. In teleost fish, the spleen (SP) and head kidney (HK) play a crucial role in peripheral immunity organs and fish skin (SK) serves as one of the most important mucosal barriers. Thus, the study focused on immune defense substances, inflammatory process, and mechanisms of molecular action in the HK, SP, and SK of on-growing grass carp (169.46–600.89 g). The fish were fed graded levels of GAA (0, 150, 300, 450 and 600 mg/kg) for 60 days, and then conducted a 14-day challenge experiment by intraperitoneal injection of Aeromonas hydrophila. In our results, we found that 300 or 450 mg/kg GAA could down-regulate the pro-inflammatory cytokines IL-(1β, 6, 12p40 and 17D), TNF-α and IFN-γ2 mRNA expression, and up-regulate the pro-inflammatory cytokines IL-(10, 11 and 4/13 A) and TGF-β1 mRNA expression in the HK, SP and SK. Furthermore, 300 or 450 mg/kg GAA could up-regulate the mRNA expression and promote the protein phosphorylation of STAT3 in the HK, SP and SK. Indicating that in the case of Aeromonas hydrophila. challenge, GAA could enhance the immune defense and mediated the inflammatory process, possibly via the JAKs-STATs signaling pathway in fish.

Anti-Inflammatory Effects of the LK5 Herbal Complex on LPS- and IL-4/IL-13-Stimulated HaCaT Cells and a DNCB-Induced Animal Model of Atopic Dermatitis in BALB/c Mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease influenced by a complex interplay of genetic and environmental factors. The activation of the JAK-STAT pathway increases the expression of inflammatory cytokines such as IL-4 and IL-13, further deteriorating AD. Therefore, for the treatment of AD, the JAK-STAT pathway is emerging as a significant target, alongside inflammatory cytokines. This study investigates the potential therapeutic effects of a novel herbal complex, LK5, composed of Scutellaria baicalensis, Liriope platyphylla, Sophora flavescens, Dictammus dasycarpus, and Phellodendron schneider, known for their anti-inflammatory and immune-modulating properties. We examined the anti-inflammatory and anti-AD effects of the LK5 herbal complex in HaCaT cells stimulated by LPS and IL-4/IL-13, as well as in a mouse model of AD induced by DNCB. In HaCaT cells stimulated with LPS or IL-4/IL-13, the LK5 herbal complex demonstrated anti-inflammatory effects by inhibiting the expression of inflammatory cytokines including TNF-α, IL-6, and IL-1β, and downregulating the phosphorylation of STAT proteins. In a murine AD-like model induced by DNCB, administration of the LK5 herbal complex significantly ameliorated clinical symptoms, including dermatitis, ear thickness, and TEWL. Histological analysis revealed a reduction in epidermal thickness and mast cell infiltration. The LK5 herbal complex also inhibited pruritus induced by compound 48/80. Furthermore, the LK5 herbal complex treatment significantly decreased the levels of inflammatory cytokines such as TSLP, IL-6, and IgE in plasma and ear tissue of AD-induced mice. These findings suggest that the LK5 herbal complex may modulate the immune response and alleviate AD symptoms by inhibiting STAT pathways.

Thymoquinone Enhances Apoptosis of K562 Chronic Myeloid Leukemia Cells through Hypomethylation of SHP-1 and Inhibition of JAK/STAT Signaling Pathway.

The epigenetic silencing of tumor suppressor genes (TSGs) is critical in the development of chronic myeloid leukemia (CML). SHP-1 functions as a TSG and negatively regulates JAK/STAT signaling. Enhancement of SHP-1 expression by demethylation provides molecular targets for the treatment of various cancers. Thymoquinone (TQ), a constituent of Nigella sativa seeds, has shown anti-cancer activities in various cancers. However, TQs effect on methylation is not fully clear. Therefore, the aim of this study is to assess TQs ability to enhance the expression of SHP-1 through modifying DNA methylation in K562 CML cells. The activities of TQ on cell cycle progression and apoptosis were evaluated using a fluorometric-red cell cycle assay and Annexin V-FITC/PI, respectively. The methylation status of SHP-1 was studied by pyrosequencing analysis. The expression of SHP-1, TET2, WT1, DNMT1, DNMT3A, and DNMT3B was determined using RT-qPCR. The protein phosphorylation of STAT3, STAT5, and JAK2 was assessed using Jess Western analysis. TQ significantly downregulated the DNMT1 gene, DNMT3A gene, and DNMT3B gene and upregulated the WT1 gene and TET2 gene. This led to hypomethylation and restoration of SHP-1 expression, resulting in inhibition of JAK/STAT signaling, induction of apoptosis, and cell cycle arrest. The observed findings imply that TQ promotes apoptosis and cell cycle arrest in CML cells by inhibiting JAK/STAT signaling via restoration of the expression of JAK/STAT-negative regulator genes.

Abstract 5026: Protein tyrosine phosphatase Ptpn1 knockout in mouse models drives B-cell hematological malignancies

Abstract Background: Ptpn1 is a member of Protein Tyrosine Phosphatase family that dephosphorylates tyrosine residues. We previously reported that an Mcm2 hypomorphic mouse (designated Mcm Cre/Cre) develops T cell acute lymphoblastic leukemia (T-ALL), due to acquisition of 100-1000 kb interstitial deletions involving tumor suppressor genes. When crossed to mice that express a NUP98-HOXD13 (NHD13) fusion gene, NHD13+;Mcm2Cre/Cre mice develop B-cell precursor ALL (BCP-ALL). Sparse whole genome sequencing revealed somatic copy number variants (CNV) involving Pax5 and Ptpn1. The role of Pax5 in B-cell development, differentiation, and leukemia has been widely studied. However, little is known about deletions of Ptpn1 and its role in BCP-ALL. Objective: The principal objective is to investigate how deletion of Ptpn1 impacts BCP-ALL development in mouse models. Methods: Mice expressing NHD13 fusion (NHD13+) were crossed with Ptpn1 knockout mice. The F2 cross generated 6 genotypes that were positive or negative for NHD13 and wild type/heterozygous/homozygous for Ptpn1. Mice were studied for incidence of BCP-ALL, which was characterized by whole-exome sequencing, molecular pathway analysis and transcriptome-sequencing. Results: NHD13+;Ptpn1-/- mice developed BCP-ALL, characterized by hyperleukocytosis, variable anemia and thrombocytopenia, expression of B220 and/or CD19, and invasion of non-hematopoietic tissues (liver, kidney, lung). In addition, NHD13+;Ptpn1+/- mice that developed BCP-ALL lost the wild-type (WT) Ptpn1 allele in the leukemic cells. Similar to human BCP-ALL, NHD13+;Ptpn1-/- mice that developed BCP-ALL showed presence of clonal TCR-delta rearrangements as well as IGH rearrangements in the leukemic cells. Whole exome sequencing showed presence of acquired mutations in genes known to be involved in B-cell differentiation, such as Pax5 or Bcor, as well as genes involved in B cell signaling pathways, such as Jak3, Jak1 and Flt3 and Western blots demonstrated accumulation of phosphorylated STAT3 protein. Conclusion: This study demonstrates that Ptpn1 loss along with expression of an NHD13 fusion gene leads to a highly penetrant BCP ALL in mice, suggesting a role for Ptpn1 in preventing malignant transformation. Taken together, these findings are consistent with a collaborative model for BCP ALL in which the NHD13 transgene leads to increased stem cell self-renewal, somatic Bcor or Pax5 mutations block normal B cell differentiation, and somatic signaling mutations (Jak1/3, Flt3) lead to hyperproliferation, which is potentiated by Ptpn1 deficiency. Citation Format: Toshihiro Matsukawa, Nupur Nigam, Ryan Bertoli, Michel L. Tremblay, Mianmian Yin, Peter D. Aplan. Protein tyrosine phosphatase Ptpn1 knockout in mouse models drives B-cell hematological malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5026.

Cytotoxic Effect of Combination Treatment of Vitamin D and Pitavastatin on the MCF7 Breast Cancer Cells

Breast cancer is the most common cancer among women. Several common approaches to cancer management are being used, but many cancer patients develop resistance to chemotherapy drugs. Here we describe the antiproliferative effect of the combination of vitamin D and pitavastatin against breast cancer cells. Aims: To determine the combined cytotoxic effect of both vitamin D and pitavastatin on the MCF7 breast cancer cell line and to study the mechanism of action behind this effect. Methods: In vitro experimental study using the MCF7 estrogen receptor-positive breast cancer cells. The effective doses of the combinations were determined by MTT, and western blotting was used to determine the different protein levels. Results: The 50% inhibitory concentrations (IC50s) of vitamin D and pitavastatin on MCF7 cells were 5.1 μM and 5.5 μM, respectively. However, when the cells were pre-treated with a low dose of vitamin D (1μM) the new pitavastatin IC50 was 1.1 μM. In addition, the combined treatment inhibited the phosphorylation of STAT3 protein in MCF7 cells and increased p53 and p21 protein levels. Conclusion: The combination of pitavastatin and vitamin D showed a synergistic cytotoxic effect by inducing cell cycle arrest and apoptosis.

STAT3 mutation-associated airway epithelial defects in Job syndrome

Job syndrome is a disease of autosomal dominant hyper-IgE syndrome (AD-HIES). Patients harboring STAT3 mutation are particularly prone to airway remodeling and airway infections. Airway epithelial cells play a central role as the first line of defense against pathogenic infection and express high levels of STAT3. This study thus interrogates how AD-HIES STAT3 mutations impact the physiological functions of airway epithelial cells. This study created human airway basal cells expressing 4 common AD-HIES STAT3 mutants (R382W, V463del, V637M, and Y657S). In addition, primary airway epithelial cells were isolated from a patient with Job syndrome who was harboring a STAT3-S560del mutation and from mice harboring a STAT3-V463del mutation. Cell proliferation, differentiation, barrier function, bacterial elimination, and innate immune responses to pathogenic infection were quantitatively analyzed. STAT3 mutations reduce STAT3 protein phosphorylation, nuclear translocation, transcription activity, and protein stability in airway basal cells. As a consequence, STAT3-mutated airway basal cells give rise to airway epithelial cells with abnormal cellular composition and loss of coordinated mucociliary clearance. Notably, AD-HIES STAT3 airway epithelial cells are defective in bacterial killing and fail to initiate vigorous proinflammatory responses and neutrophil transepithelial migration in response to an experimental model of Pseudomonas aeruginosa infection. AD-HIES STAT3 mutations confer numerous abnormalities to airway epithelial cells in cell differentiation and host innate immunity, emphasizing their involvement in the pathogenesis of lung complications in Job syndrome. Therefore, therapies must address the epithelial defects as well as the previously noted immune cell defects to alleviate chronic infections in patients with Job syndrome.

Fibrotic and inflammatory characteristics of epidural fat adjacent to the ossification area in patients with ossification of the ligament flavum.

To observe histological and inflammatory characteristics of epidural fat (EF) adjacent to the ossification area in patients with ossification of the thoracic ligament flavum (TOLF) and provide a preliminary research basis for investigating the impact of the EF on OLF. Samples of EF and autologous subcutaneous adipose tissue (SCAT) from TOLF patients (n=26) and non-TOLF patients (n=23) were harvested during posterior thoracic spine surgery. Adipocyte size and fibrosis were measured by histology. Vascularization and inflammatory cell infiltration were evaluated by immunohistochemical staining. Adipogenesis-related genes were assessed by real-time quantitative PCR. Conditioned media from cultured EF were evaluated via enzyme-linked immunosorbent assay to detect the secretion of inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and leptin. The phosphorylated STAT3 protein level in ligament flavum (LF) was examined using Western blot. Adipocytes size in EF was similar between in the TOLF and non-TOLF groups, but significantly smaller than that from autologous SCAT. Adipogenesis-related mRNA expression in EF was lower than that in SCAT. More fibrosis and vascularization were found in TOLF than in non-TOLF. EF in the TOLF group exhibited more macrophages and B lymphocytes infiltrated. The levels of cytokines such as IL-6, TNF-α, and leptin secreted by EF were significantly higher in the TOLF group than non-TOLF. The level of phosphorylated STAT3 in LF was significantly upregulated in the TOLF group. Morphologically, EF adjacent to the ossification area is smaller and more uniform than autologous SCAT, exhibiting a characteristic similarity to visceral fat. EF in the TOLF group shows a more fibrotic, vascularized, and inflammatory phenotype, which secretes multiple cytokines. The phosphorylated STAT3 protein was significantly upregulated in the TOLF group. Whether these properties of EF directly affect the process of OLF needs to be further explored.

Cytokines Activate JAK-STAT Signaling Pathway in MG-63 Cells on Titanium and Zirconia.

Although titanium has been traditionally used as the gold standard for dental implants, recent years have seen the widespread application of zirconia implants given their superiority with regards to reduced bacterial adhesion, inflammation and cellular-interaction in terms of bio-compatibility. The JAK–STAT signaling pathway plays an important role in bone remodeling and formation. The aim of the study was to investigate the activation of the JAK–STAT pathway through different cytokines in osteoblast-like cells (MG-63) on zirconia in comparison to titanium discs. IFN-γ induced the very strong activation of STAT1 protein, IFN-α activated both STAT1 and STAT3 molecules, IL-6 activated STAT3 and IL-4 induced the activation of STAT6 on both surfaces. The activation of STAT proteins was confirmed by western blot, immunofluorescence and flow cytometry using phospho-specific anti-STAT antibodies, which recognize only phosphorylated STAT proteins. The incubation of MG-63 cells with IFN-γ caused the upregulation of MHC class I and class II proteins when MG-63 cells were grown on zirconia and titanium discs. In sum, the present study shows that the JAK–STAT pathway is activated in MG-63 cells when they are incubated on titanium or zirconia surfaces.

The miR-590-3p/CFHR3/STAT3 signaling pathway promotes cell proliferation and metastasis in hepatocellular carcinoma.

Accumulating evidence has indicated that Complement factor H-related 3 (CFHR3) plays an essential role in various diseases. However, the biological functions of CFHR3 in hepatocellular carcinoma (HCC) remain largely unclear. Therefore, we perform a further study on CFHR3 in HCC. In this article, we report the suppressive role of CFHR3 in the proliferation and metastasis of HCC cells. CFHR3 downregulation is closely associated with large (T3-T4) HCC, tumor recurrence, and advanced (stage III-IV) clinical stage, functioning as an independent factor for the prognoses of HCC patients. Knockdown of CFHR3 promotes proliferation, migration, and invasion of HCC cells. Mechanistically, downregulation of CFHR3 is induced by miR-590-3p binding to the 3’ untranslated region (UTR) of CFHR3. CFHR3 downregulation promotes the phosphorylation of STAT3 protein, thereby suppressing p53 expression. The promotional effect upon downregulation of CFHR3 induced by CFHR3 stable knockdown or miR-590-3p on HCC cell malignant phenotypes is attenuated by STAT3 inhibitor, S3I-201. In conclusion, our results reveal that CFHR3 is a protective biomarker for HCC patients, and targeting the miR-590-3p/CFHR3/p-STAT3/p53 signaling axis provides a promising strategy for HCC therapeutics.

The Immune Profile of Major Dysmood Disorder: Proof of Concept and Mechanism Using the Precision Nomothetic Psychiatry Approach.

Major depressive disorder and a major depressive episode (MDD/MDE) are characterized by activation of the immune-inflammatory response system (IRS) and the compensatory immune-regulatory system (CIRS). In MDD/MDE, recent precision nomothetic psychiatry studies discovered a new endophenotype class, namely major dysmood disorder (MDMD), a new pathway phenotype, namely reoccurrence of illness (ROI), and a new model of the phenome of depression. The aim of the present study is to examine the association between ROI, the phenome of depression, and MDMD’s features and IRS, CIRS, macrophages (M1), T helper (Th)1, Th2, Th17, T regulatory, and growth factor (GF) profiles. Culture supernatants of unstimulated and stimulated (5 μg/mL of PHA and 25 μg/mL of LPS) diluted whole blood of 30 MDD/MDE patients and 20 controls were assayed for cytokines/GF using the LUMINEX assay. MDMD was characterized by increased M1, Th1, Th2, Th17, Treg, IRS, CIRS, neurotoxicity, and GF profiles. Factor analysis shows that ROI features and immune-GF profiles may be combined into a new pathway phenotype (an extracted latent vector). ROI, lifetime and recent suicidal behaviors, and severity of depression are significantly associated with immunotoxicity and GF profiles. Around 80.0% of the variance in the phenome is predicted by ROI and neurotoxicity or the IRS/CIRS ratio. The molecular pathways underpinning ROI-associated sensitization of immune/growth networks are transmembrane receptor protein kinase-triggered STAT protein phosphorylation, TLR/NF-κB, JAK-STAT, and the main proliferation/survival PI3K/Akt/RAS/MAPK pathway. In conclusion, MDMD’s heightened immune responses are the consequence of ROI-associated sensitization combined with immunostimulatory triggers.

The Janus kinase inhibitor (baricitinib) suppresses the rheumatoid arthritis active marker gliostatin/thymidine phosphorylase in human fibroblast-like synoviocytes

Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities in the pathogenesis of rheumatoid arthritis (RA). The novel oral Janus kinase (JAK) inhibitor baricitinib has demonstrated high efficacy in RA. However, the effect of baricitinib on fibroblast-like synoviocytes (FLSs), a key component of invasive synovitis, has not been still elucidated. This study investigated whether GLS/TP production could be regulated by JAK/signal transducers and activators of transcription (STAT) signaling in FLSs derived from patients with RA. FLSs were cultured and stimulated by interferon (IFN)γ in the presence of baricitinib. Expression levels of GLS/TP were determined using reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunocytochemistry. Phosphorylation of STAT proteins was investigated by Western blot. In cultured FLSs, GLS/TP mRNA and protein levels were significantly induced by treatment with IFNγ and these inductions were suppressed by baricitinib treatment. Baricitinib inhibited IFNγ-induced STAT1 phosphorylation, while JAK/STAT activation played a pivotal role in IFNγ-mediated GLS/TP upregulation in RA. These results suggested that baricitinib suppressed IFNγ-induced GLS/TP expression by inhibiting JAK/STAT signaling, resulting in the attenuation of neovascularization, synovial inflammation, and cartilage destruction.

Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

IFN-γ inhibits ovarian cancer progression via SOCS1/JAK/STAT signaling pathway.

Ovarian cancer (OC) is a common malignancy, and IFN-γ, a multifunctional cytokine, is unveiled to impede the multiplication and enhance apoptosis in diverse tumor cells in previous research. Nonetheless, its function and mechanism in OC are blurred. OC cell lines SKOV3 and OVCAR3 were dealt with different concentrations (0-40ng/ml) of IFN-γ. CCK-8 experiment was utilized to examine cell multiplication; Flow cytometry was executed to detect apoptosis and cell cycle; Wound healing assay was utilized to detect cell migration; and Transwell experiment was implemented to examine cell invasion. qRT-PCR analysis was applied to detect STAT5, STAT3, JAK2 and JAK3 mRNA expression in OC cell lines. Western blot experiment was applied to detect the protein and phosphorylation levels of SOCS1, STAT5 and STAT3. IFN-γ suppressed OC cell multiplication in a concentration-dependent manner. Relative to the control group, IFN-γ restrained OC cell migration, invasion, enhanced apoptosis and prevented cell transformation from G0/G1 to S phase. Further analysis revealed that IFN-γ up-modulated SOCS1 expression and impeded STAT5 and STAT3 protein phosphorylation levels, and knockdown of SOCS1 partially counteracted the inhibitory effect of IFN-γ on STAT5 and STAT3 protein phosphorylation levels. IFN-γ represses OC progression by facilitating SOCS1 to suppress STAT3 and STAT5 protein phosphorylation.

Abstract 2000: Different oncogenic KRAS mutations produce distinct heterogenous outcomes in signaling pathways of isogenic mouse embryonic fibroblasts

Abstract Background: Growing evidence points towards the concept that not all RAS allelic variances are equal. Different RAS mutations produce distinct variations in signal transduction to sustain their proliferative needs accordingly. This is an especially important concept to consider as medicine shifts towards a more personalized approach. In accordance with this notion, through this study, we aim to explore the network dynamics and pinpoint disparities in signaling events associated with seven common KRAS allelic variances in non-small cell lung carcinoma. Methods: We used Mouse Embryonic Fibroblasts (MEF) provided by the RAS initiative (https://www.cancer.gov/research/key-initiatives/ras/outreach/reference-reagents/cell-lines). MEF were engineered to selectively harbor and express either wild type KRAS (4A and 4B WT) or one of seven following mutations: 4B G12D, G12C, G12R, G12V, G13D, Q61R and Q61L. Using high throughput immunoassay, Reverse Phase Protein Microarray (RPPA), we explored broad changes in signaling dynamics across our models. Specifically, we captured expression and/or activation levels of 68 proteins and phosphoproteins including: Receptor Tyrosine Kinases, MAPK, AKT/mTOR, JNK/STAT. Data were analyzed using unsupervised hierarchical clustering analysis using the Ward methods and the Kruskal Wallis test. P values <0.05 were considered significant. Results: Based on the unsupervised clustering analysis, Q61R and Q61L mutations showed the least activated signaling when compared to all other models. Activation of Raf was increased in cells harboring mutations of the G12 residues compared to those with mutations of the G13 and Q61 residues. Furthermore, the five G12 mutations clustered together with an overall higher active signaling compared to other cell lines. However, among the group of G12 mutants, unique signal transduction profiles were identified. We report distinct significant differences that divide the clustered G12 mutants into high versus low activation of specific signaling proteins. For example, ERK T202/Y204 (p=0.011) and S6 Ribosomal Protein S235/236 (p=0.001) phosphorylation were significantly higher in G12R compared to G12C and G12D models. On the contrary, STAT1 and STAT3 protein phosphorylation at residues Y701 and Y705 respectively were decreased in the G12R model. G12C has the lowest activation levels of p90RSK S380 (p=0.029), NF-KappaB p65 S536 (p=0.027) and P70 S6 Kinase S371 (p=0.013). Conclusions: In alignment with other findings, we show that based on RAS mutation, individual signaling components are affected differently, producing extensive heterogeneity in signaling dynamics. Even within grouped mutations presenting an overall similar net effect on downstream signaling, each individual variant produced discrete alterations. Citation Format: Emna El Gazzah, Elisa Baldelli, Chamodya Ruhunsiri, Emanuel F. Petricoin, Mariaelena Pierobon. Different oncogenic KRAS mutations produce distinct heterogenous outcomes in signaling pathways of isogenic mouse embryonic fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2000.

Metformin ameliorates scleroderma via inhibiting Th17 cells and reducing mTOR-STAT3 signaling in skin fibroblasts

Scleroderma is an autoimmune disease that causes dermal fibrosis. It occurs when collagen accumulates in tissue as a result of persistent inflammation. Th17 cells and pro-inflammatory cytokines such as IL-1β, IL-6, IL-17, and TNF-α play important roles in the pathogenesis of scleroderma. Because metformin, a medication used to treat diabetes, has effective immunoregulatory functions, we investigated its therapeutic function in scleroderma. Mice in a model of bleomycin-induced scleroderma were treated with metformin for 2 weeks. Histological assessment demonstrated protective effects of metformin against scleroderma. Metformin decreased the expression of pro-inflammatory factors in dermal tissue and lymphocytes. It also decreased mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, IL-17, and TNF-α) and fibrosis-inducing molecules both in vivo and in vitro. These results suggest that metformin treatment has anti-inflammatory effects on lymphocytes via the inhibition of IL-17 and cytokines related to Th17 differentiation, such as IL-1β, IL-6, and TNF-α. To investigate how metformin modulates the inflammatory process in skin fibroblasts, we measured mTOR-STAT3 signaling in skin fibroblasts and found that phosphorylated mTOR and phosphorylated STAT3 protein expression were decreased by metformin treatment. These results suggest that metformin has potential to treat scleroderma by inhibiting pro-inflammatory cytokines and anti-inflammatory activity mediated by mTOR-STAT3 signaling.