Signal transducer and activator of transcription 3 (STAT3) signalling serves an important role in carcinogenesis and cellular senescence, and its inhibition in tumour cells represents an attractive therapeutic target. Premature cellular senescence, a process of permanent proliferative arrest of cells in response to various inducers, such as cytostatic drugs or ionizing radiation, is accompanied by morphological and secretory changes, and by altered susceptibility to chemotherapeutic agents, which can thereby complicate their eradication by cancer therapies. In the present study, the responsiveness of proliferating and docetaxel (DTX)‑induced senescent cancer cells to small molecule STAT3 inhibitor Stattic and its analogues was evaluated using tumour cell lines. These agents displayed cytotoxic effects in cell viability assays on both proliferating and senescent murine TRAMP‑C2 and TC‑1 cells; however, senescent cells were markedly more resistant. Western blot analysis revealed that Stattic and its analogues effectively inhibited constitutive STAT3 phosphorylation in both proliferating and senescent cells. Furthermore, whether the Stattic‑derived inhibitor K1836 could affect senescence induction or modulate the phenotype of senescent cells was evaluated. K1836 treatment demonstrated no effect on senescence induction by DTX. However, the K1836 compound significantly modulated secretion of certain cytokines (interleukin‑6, growth‑regulated oncogene α and monocyte chemoattractant protein‑1). In summary, the present study demonstrated differences between proliferating and senescent tumour cells in terms of their susceptibility to STAT3 inhibitors and demonstrated the ability of the new STAT3 inhibitor K1836 to affect the secretion of essential components of the senescence‑associated secretory phenotype. The present study may be useful for further development of STAT3 inhibitor‑based therapy of cancer or age‑related diseases.

• Keywords
• Stattic, cellular senescence, docetaxel, senescence‑associated secretory phenotype, signal transducer and activator of transcription 3 inhibition,
• MeSH
• Cytokines * metabolism   MeSH
• Docetaxel pharmacology   MeSH
• Gene Expression MeSH
• Phosphorylation MeSH
• Mice MeSH
• Cellular Senescence MeSH
• STAT3 Transcription Factor * metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytokines * MeSH
• Docetaxel MeSH
• stattic MeSH Browser
• STAT3 Transcription Factor * MeSH

Patients with lower-risk myelodysplastic syndromes (LR-MDS) have a generally favorable prognosis; however, a small proportion of cases progress rapidly. This study aimed to define molecular biomarkers predictive of LR-MDS progression and to uncover cellular pathways contributing to malignant transformation. The mutational landscape was analyzed in 214 LR-MDS patients, and at least one mutation was detected in 137 patients (64%). Mutated RUNX1 was identified as the main molecular predictor of rapid progression by statistics and machine learning. To study the effect of mutated RUNX1 on pathway regulation, the expression profiles of CD34 + cells from LR-MDS patients with RUNX1 mutations were compared to those from patients without RUNX1 mutations. The data suggest that RUNX1-unmutated LR-MDS cells are protected by DNA damage response (DDR) mechanisms and cellular senescence as an antitumor cellular barrier, while RUNX1 mutations may be one of the triggers of malignant transformation. Dysregulated DDR and cellular senescence were also observed at the functional level by detecting γH2AX expression and β-galactosidase activity. Notably, the expression profiles of RUNX1-mutated LR-MDS resembled those of higher-risk MDS at diagnosis. This study demonstrates that incorporating molecular data improves LR-MDS risk stratification and that mutated RUNX1 is associated with a suppressed defense against LR-MDS progression.

• MeSH
• Leukemia, Myeloid, Acute * genetics   MeSH
• Humans MeSH
• Mutation MeSH
• Myelodysplastic Syndromes * pathology   MeSH
• Cell Transformation, Neoplastic genetics   metabolism   MeSH
• Prognosis MeSH
• Core Binding Factor Alpha 2 Subunit genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Core Binding Factor Alpha 2 Subunit MeSH
• RUNX1 protein, human MeSH Browser

Cellular senescence is a complex stress response defined as an essentially irreversible cell cycle arrest mediated by the inhibition of cell cycle-specific cyclin dependent kinases. The imbalance in redox homeostasis and oxidative stress have been repeatedly observed as one of the hallmarks of the senescent phenotype. However, a large-scale study investigating protein oxidation and redox signaling in senescent cells in vitro has been lacking. Here we applied a proteome-wide analysis using SILAC-iodoTMT workflow to quantitatively estimate the level of protein sulfhydryl oxidation and proteome level changes in ionizing radiation-induced senescence (IRIS) in hTERT-RPE-1 cells. We observed that senescent cells mobilized the antioxidant system to buffer the increased oxidation stress. Among the antioxidant proteins with increased relative abundance in IRIS, a unique 1-Cys peroxiredoxin family member, peroxiredoxin 6 (PRDX6), was identified as an important contributor to protection against oxidative stress. PRDX6 silencing increased ROS production in senescent cells, decreased their resistance to oxidative stress-induced cell death, and impaired their viability. Subsequent SILAC-iodoTMT and secretome analysis after PRDX6 silencing showed the downregulation of PRDX6 in IRIS affected protein secretory pathways, decreased expression of extracellular matrix proteins, and led to unexpected attenuation of senescence-associated secretory phenotype (SASP). The latter was exemplified by decreased secretion of pro-inflammatory cytokine IL-6 which was also confirmed after treatment with an inhibitor of PRDX6 iPLA2 activity, MJ33. In conclusion, by combining different methodological approaches we discovered a novel role of PRDX6 in senescent cell viability and SASP development. Our results suggest PRDX6 could have a potential as a drug target for senolytic or senomodulatory therapy.

• Keywords
• Cellular senescence, Interleukin 6, Peroxiredoxin 6, Redox proteomics, SILAC-iodoTMT, Senescence-associated secretory phenotype,
• MeSH
• Cytokines * metabolism   MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress MeSH
• Peroxiredoxin VI * genetics   metabolism   MeSH
• Cellular Senescence physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines * MeSH
• Peroxiredoxin VI * MeSH

Molecular pathophysiology of Diamond-Blackfan anemia (DBA) involves disrupted erythroid-lineage proliferation, differentiation and apoptosis; with the activation of p53 considered as a key component. Recently, oxidative stress was proposed to play an important role in DBA pathophysiology as well. CRISPR/Cas9-created Rpl5- and Rps19-deficient murine erythroleukemia (MEL) cells and DBA patients' samples were used to evaluate proinflammatory cytokines, oxidative stress, DNA damage and DNA damage response. We demonstrated that the antioxidant defense capacity of Rp-mutant cells is insufficient to meet the greater reactive oxygen species (ROS) production which leads to oxidative DNA damage, cellular senescence and activation of DNA damage response signaling in the developing erythroblasts and altered characteristics of mature erythrocytes. We also showed that the disturbed balance between ROS formation and antioxidant defense is accompanied by the upregulation of proinflammatory cytokines. Finally, the alterations detected in the membrane of DBA erythrocytes may cause their enhanced recognition and destruction by reticuloendothelial macrophages, especially during infections. We propose that the extent of oxidative stress and the ability to activate antioxidant defense systems may contribute to high heterogeneity of clinical symptoms and response to therapy observed in DBA patients.

• Keywords
• 8-oxoguanine, DNA damage response, Diamond-Blackfan anemia, erythrocyte lifespan, inflammatory cytokines, reactive oxygen species,
• MeSH
• Anemia, Diamond-Blackfan immunology   metabolism   pathology   MeSH
• Child MeSH
• Adult MeSH
• Erythrocytes metabolism   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Inflammation Mediators metabolism   MeSH
• Young Adult MeSH
• Mice MeSH
• Follow-Up Studies MeSH
• Oxidative Stress * MeSH
• DNA Damage * MeSH
• Prognosis MeSH
• Case-Control Studies MeSH
• Inflammation immunology   metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Inflammation Mediators MeSH

Inflammatory and oncogenic signaling converge in disease evolution of BCR-ABL-negative myeloproliferative neoplasms, clonal hematopoietic stem cell disorders characterized by gain-of-function mutation in JAK2 kinase (JAK2V617F), with highest prevalence in patients with polycythemia vera (PV). Despite the high risk, DNA-damaging inflammatory microenvironment, PV progenitors tend to preserve their genomic stability over decades until their progression to post-PV myelofibrosis/acute myeloid leukemia. Using induced pluripotent stem cells-derived CD34+ progenitor-enriched cultures from JAK2V617F+ PV patient and from JAK2 wild-type healthy control, CRISPR-modified HEL cells and patients' bone marrow sections from different disease stages, we demonstrate that JAK2V617F induces an intrinsic IFNγ- and NF-κB-associated inflammatory program, while suppressing inflammation-evoked DNA damage both in vitro and in vivo. We show that cells with JAK2V617F tightly regulate levels of inflammatory cytokines-induced reactive oxygen species, do not fully activate the ATM/p53/p21waf1 checkpoint and p38/JNK MAPK stress pathway signaling when exposed to inflammatory cytokines, suppress DNA single-strand break repair genes' expression yet overexpress the dual-specificity phosphatase (DUSP) 1. RNAi-mediated knock-down and pharmacological inhibition of DUSP1, involved in p38/JNK deactivation, in HEL cells reveals growth addiction to DUSP1, consistent with enhanced DNA damage response and apoptosis in DUSP1-inhibited parental JAK2V617F+ cells, but not in CRISPR-modified JAK2 wild-type cells. Our results indicate that the JAK2V617F+ PV progenitors utilize DUSP1 activity as a protection mechanism against DNA damage accumulation, promoting their proliferation and survival in the inflammatory microenvironment, identifying DUSP1 as a potential therapeutic target in PV.

• MeSH
• Cytokines genetics   metabolism   MeSH
• Dual Specificity Phosphatase 1 genetics   MeSH
• Hematopoietic Stem Cells pathology   MeSH
• Induced Pluripotent Stem Cells pathology   MeSH
• Janus Kinase 2 genetics   MeSH
• Humans MeSH
• Mutation MeSH
• Cell Line, Tumor MeSH
• Tumor Microenvironment MeSH
• Oxidative Stress * MeSH
• Polycythemia Vera genetics   MeSH
• DNA Damage * MeSH
• Cell Proliferation * MeSH
• Reproducibility of Results MeSH
• STAT1 Transcription Factor metabolism   MeSH
• Inflammation metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• DUSP1 protein, human MeSH Browser
• Dual Specificity Phosphatase 1 MeSH
• JAK2 protein, human MeSH Browser
• Janus Kinase 2 MeSH
• STAT1 protein, human MeSH Browser
• STAT1 Transcription Factor MeSH

Cellular senescence is a form of cell cycle arrest that limits the proliferative potential of cells, including tumour cells. However, inability of immune cells to subsequently eliminate senescent cells from the organism may lead to tissue damage, inflammation, enhanced carcinogenesis and development of age-related diseases. We found that the anticancer agent mitochondria-targeted tamoxifen (MitoTam), unlike conventional anticancer agents, kills cancer cells without inducing senescence in vitro and in vivo. Surprisingly, it also selectively eliminates both malignant and non-cancerous senescent cells. In naturally aged mice treated with MitoTam for 4 weeks, we observed a significant decrease of senescence markers in all tested organs compared to non-treated animals. Mechanistically, we found that the susceptibility of senescent cells to MitoTam is linked to a very low expression level of adenine nucleotide translocase-2 (ANT2), inherent to the senescent phenotype. Restoration of ANT2 in senescent cells resulted in resistance to MitoTam, while its downregulation in non-senescent cells promoted their MitoTam-triggered elimination. Our study documents a novel, translationally intriguing role for an anticancer agent targeting mitochondria, that may result in a new strategy for the treatment of age-related diseases and senescence-associated pathologies.

• MeSH
• Apoptosis drug effects   genetics   MeSH
• Gene Knockdown Techniques MeSH
• Antineoplastic Agents, Hormonal pharmacology   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• Mitochondria drug effects   metabolism   MeSH
• Mice, Inbred NOD MeSH
• Mice, SCID MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Cell Proliferation drug effects   MeSH
• Cellular Senescence drug effects   MeSH
• Tamoxifen pharmacology   MeSH
• Transfection MeSH
• Adenine Nucleotide Translocator 2 genetics   metabolism   MeSH
• Cell Survival drug effects   genetics   MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antineoplastic Agents, Hormonal MeSH
• Tamoxifen MeSH
• Adenine Nucleotide Translocator 2 MeSH

Cellular senescence is the process of the permanent proliferative arrest of cells in response to various inducers. It is accompanied by typical morphological changes, in addition to the secretion of bioactive molecules, including proinflammatory cytokines and chemokines [known as the senescence-associated secretory phenotype (SASP)]. Thus, senescent cells may affect their local environment and induce a so-called 'bystander' senescence through the state of SASP. The phenotypes of senescent cells are determined by the type of agent inducing cellular stress and the cell lineages. To characterise the phenotypes of senescent cancer cells, two murine cell lines were employed in the present study: TC-1 and B16F10 (B16) cells. Two distinct senescence inductors were used: Chemotherapeutic agent docetaxel (DTX) and a combination of immunomodulatory cytokines, including interferon γ (IFNγ) and tumour necrosis factor α (TNFα). It was demonstrated that DTX induced senescence in TC-1 and B16 tumour cell lines, which was demonstrated by growth arrest, positive β-galactosidase staining, increased p21Waf1 (p21) expression and the typical SASP capable of inducing a 'bystander' senescence. By contrast, treatment with a combination of T helper cell 1 cytokines, IFNγ and TNFα, induced proliferation arrest only in B16 cells. Despite the presence of certain characteristic features resembling senescent cells (proliferation arrest, morphological changes and increased p21 expression), these cells were able to form tumours in vivo and started to proliferate upon cytokine withdrawal. In addition, B16 cells were not able to induce a 'bystander' senescence. In summary, the present study described cell line- and treatment-associated differences in the phenotypes of senescent cells that may be relevant in optimization of cancer chemo- and immunotherapy.

• MeSH
• Bystander Effect drug effects   immunology   MeSH
• Docetaxel pharmacology   therapeutic use   MeSH
• Phenotype MeSH
• Interferon-gamma immunology   metabolism   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Neoplasms drug therapy   immunology   pathology   MeSH
• Cell Proliferation drug effects   MeSH
• Antineoplastic Agents pharmacology   therapeutic use   MeSH
• Cellular Senescence drug effects   immunology   MeSH
• Tumor Necrosis Factor-alpha immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Docetaxel MeSH
• IFNG protein, mouse MeSH Browser
• Interferon-gamma MeSH
• Antineoplastic Agents MeSH
• Tumor Necrosis Factor-alpha MeSH

Aging involves tissue accumulation of senescent cells (SC) whose elimination through senolytic approaches may evoke organismal rejuvenation. SC also contribute to aging-associated pathologies including cancer, hence it is imperative to better identify and target SC. Here, we aimed to identify new cell-surface proteins differentially expressed on human SC. Besides previously reported proteins enriched on SC, we identified 78 proteins enriched and 73 proteins underrepresented in replicatively senescent BJ fibroblasts, including L1CAM, whose expression is normally restricted to the neural system and kidneys. L1CAM was: 1) induced in premature forms of cellular senescence triggered chemically and by gamma-radiation, but not in Ras-induced senescence; 2) induced upon inhibition of cyclin-dependent kinases by p16INK4a; 3) induced by TGFbeta and suppressed by RAS/MAPK(Erk) signaling (the latter explaining the lack of L1CAM induction in RAS-induced senescence); and 4) induced upon downregulation of growth-associated gene ANT2, growth in low-glucose medium or inhibition of the mevalonate pathway. These data indicate that L1CAM is controlled by a number of cell growth- and metabolism-related pathways during SC development. Functionally, SC with enhanced surface L1CAM showed increased adhesion to extracellular matrix and migrated faster. Our results provide mechanistic insights into senescence of human cells, with implications for future senolytic strategies.

• Keywords
• MAPK pathway, SILAC, aging, mass spectrometry, proteomics,
• MeSH
• Cell Adhesion physiology   MeSH
• Cell Cycle MeSH
• Down-Regulation MeSH
• Fibroblasts MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Humans MeSH
• Neural Cell Adhesion Molecule L1 genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Cell Movement physiology   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Gene Expression Regulation drug effects   radiation effects   MeSH
• RNA Interference MeSH
• Signal Transduction MeSH
• Cellular Senescence MeSH
• Transforming Growth Factor beta metabolism   pharmacology   MeSH
• Gamma Rays MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Neural Cell Adhesion Molecule L1 MeSH
• Transforming Growth Factor beta MeSH

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis frequently progressing into acute myeloid leukemia (AML), with emerging evidence implicating aberrant bone marrow (BM) microenvironment and inflammation-related changes. 5-azacytidine (5-AC) represents standard MDS treatment. Besides inhibiting DNA/RNA methylation, 5-AC has been shown to induce DNA damage and apoptosis in vitro. To provide insights into in vivo effects, we assessed the proinflammatory cytokines alterations during MDS progression, cytokine changes after 5-AC, and contribution of inflammatory comorbidities to the cytokine changes in MDS patients. We found that IL8, IP10/CXCL10, MCP1/CCL2 and IL27 were significantly elevated and IL12p70 decreased in BM of MDS low-risk, high-risk and AML patients compared to healthy donors. Repeated sampling of the high-risk MDS patients undergoing 5-AC therapy revealed that the levels of IL8, IL27 and MCP1 in BM plasma were progressively increasing in agreement with in vitro experiments using several cancer cell lines. Moreover, the presence of inflammatory diseases correlated with higher levels of IL8 and MCP1 in low-risk but not in high-risk MDS. Overall, all forms of MDS feature a deregulated proinflammatory cytokine landscape in the BM and such alterations are further augmented by therapy of MDS patients with 5-AC.

• Keywords
• 5-azacytidine, DNA damage, bone marrow plasma, cytokines, inflammation, myelodysplastic syndromes,
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cellular senescence provides a biological barrier against tumor progression, often associated with oncogene-induced replication and/or oxidative stress, cytokine production and DNA damage response (DDR), leading to persistent cell-cycle arrest. While cytokines such as tumor necrosis factor-alpha (TNFα) and interferon gamma (IFNγ) are important components of senescence-associated secretome and induce senescence in, for example, mouse pancreatic β-cancer cell model, their downstream signaling pathway(s) and links with oxidative stress and DDR are mechanistically unclear. Using human and mouse normal and cancer cell models, we now show that TNFα and IFNγ induce NADPH oxidases Nox4 and Nox1, reactive oxygen species (ROS), DDR signaling and premature senescence. Unlike mouse tumor cells that required concomitant presence of IFNγ and TNFα, short exposure to IFNγ alone was sufficient to induce Nox4, Nox1 and DDR in human cells. siRNA-mediated knockdown of Nox4 but not Nox1 decreased IFNγ-induced DDR. The expression of Nox4/Nox1 required Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling and the effect was mediated by downstream activation of transforming growth factor-beta (TGFβ) secretion and consequent autocrine/paracrine activation of the TGFβ/Smad pathway. Furthermore, the expression of adenine nucleotide translocase 2 (ANT2) was suppressed by IFNγ contributing to elevation of ROS and DNA damage. In contrast to mouse B16 cells, inability of TC-1 cells to respond to IFNγ/TNFα by DDR and senescence correlated with the lack of TGFβ and Nox4 response, supporting the role of ROS induced by NADPH oxidases in cytokine-induced senescence. Overall, our data reveal differences between cytokine effects in mouse and human cells, and mechanistically implicate the TGFβ/SMAD pathway, via induction of NADPH oxidases and suppression of ANT2, as key mediators of IFNγ/TNFα-evoked genotoxicity and cellular senescence.

• MeSH
• Enzyme Induction drug effects   MeSH
• Interferon-gamma pharmacology   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• NADPH Oxidase 1 MeSH
• NADPH Oxidase 4 MeSH
• NADPH Oxidases biosynthesis   genetics   MeSH
• Oxidative Stress drug effects   MeSH
• DNA Damage * MeSH
• Smad Proteins metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Signal Transduction drug effects   MeSH
• Cellular Senescence drug effects   MeSH
• Tumor Necrosis Factor-alpha pharmacology   MeSH
• Transforming Growth Factor beta metabolism   MeSH
• STAT Transcription Factors metabolism   MeSH
• Adenine Nucleotide Translocator 2 metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interferon-gamma MeSH
• NADPH Oxidase 1 MeSH
• NADPH Oxidase 4 MeSH
• NADPH Oxidases MeSH
• NOX1 protein, human MeSH Browser
• NOX4 protein, human MeSH Browser
• Smad Proteins MeSH
• Reactive Oxygen Species MeSH
• Tumor Necrosis Factor-alpha MeSH
• Transforming Growth Factor beta MeSH
• STAT Transcription Factors MeSH
• Adenine Nucleotide Translocator 2 MeSH