p53-mutated tumors often exhibit increased resistance to standard chemotherapy and enhanced metastatic potential. Here we demonstrate that inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme of the de novo pyrimidine synthesis pathway, effectively decreases proliferation of cancer cells via induction of replication and ribosomal stress in a p53- and checkpoint kinase 1 (Chk1)-dependent manner. Mechanistically, a block in replication and ribosomal biogenesis result in p53 activation paralleled by accumulation of replication forks that activate the ataxia telangiectasia and Rad3-related kinase/Chk1 pathway, both of which lead to cell cycle arrest. Since in the absence of functional p53 the cell cycle arrest fully depends on Chk1, combined DHODH/Chk1 inhibition in p53-dysfunctional cancer cells induces aberrant cell cycle re-entry and erroneous mitosis, resulting in massive cell death. Combined DHODH/Chk1 inhibition effectively suppresses p53-mutated tumors and their metastasis, and therefore presents a promising therapeutic strategy for p53-mutated cancers.
- MeSH
- Checkpoint Kinase 1 antagonists & inhibitors genetics metabolism MeSH
- Phenylurea Compounds pharmacology MeSH
- Genes, erbB-2 MeSH
- HCT116 Cells MeSH
- Protein Kinase Inhibitors pharmacology MeSH
- Cell Cycle Checkpoints * drug effects MeSH
- Leflunomide pharmacology MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Mice, Inbred BALB C MeSH
- Mice, Inbred NOD MeSH
- Mice, SCID MeSH
- Mice, Transgenic MeSH
- Tumor Suppressor Protein p53 deficiency genetics MeSH
- Breast Neoplasms drug therapy genetics metabolism pathology MeSH
- Oxidoreductases Acting on CH-CH Group Donors antagonists & inhibitors genetics metabolism MeSH
- Cell Proliferation * drug effects MeSH
- Antineoplastic Combined Chemotherapy Protocols pharmacology MeSH
- Pyrazines pharmacology MeSH
- Pyrimidines biosynthesis MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Ribosomes genetics metabolism MeSH
- Signal Transduction MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Radiation and chemotherapy represent standard-of-care cancer treatments. However, most patients eventually experience tumour recurrence, treatment failure and metastatic dissemination with fatal consequences. To elucidate the molecular mechanisms of resistance to radio- and chemotherapy, we exposed human cancer cell lines (HeLa, MCF-7 and DU145) to clinically relevant doses of 5-azacytidine or ionizing radiation and compared the transcript profiles of all surviving cell subpopulations, including low-adherent stem-like cells. Stress-mobilized low-adherent cell fractions differed from other survivors in terms of deregulation of hundreds of genes, including those involved in interferon response. Exposure of cancer cells to interferon-gamma but not interferon-beta resulted in the development of a heterogeneous, low-adherent fraction comprising not only apoptotic/necrotic cells but also live cells exhibiting active Notch signalling and expressing stem-cell markers. Chemical inhibition of mitogen-activated protein kinase/ERK kinase (MEK) or siRNA-mediated knockdown of extracellular signal-regulated kinase 1/2 (Erk1/2) and interferon responsible factor 1 (IRF1) prevented mobilization of the surviving low-adherent population, indicating that interferon-gamma-mediated loss of adhesion and anoikis resistance required an active Erk pathway interlinked with interferon signalling by transcription factor IRF1. Notably, a skin-specific protein suprabasin (SBSN), a recently identified oncoprotein, was among the top scoring genes upregulated in surviving low-adherent cancer cells induced by 5-azacytidine or irradiation. SBSN expression required the activity of the MEK/Erk pathway, and siRNA-mediated knockdown of SBSN suppressed the low-adherent fraction in irradiated, interferon-gamma- and 5-azacytidine-treated cells, respectively, implicating SBSN in genotoxic stress-induced phenotypic plasticity and stress resistance. Importantly, SBSN expression was observed in human clinical specimens of colon and ovarian carcinomas, as well as in circulating tumour cells and metastases of the 4T1 mouse model. The association of SBSN expression with progressive stages of cancer development indicates its role in cancer evolution and therapy resistance.
- MeSH
- Anoikis drug effects radiation effects MeSH
- Azacitidine pharmacology MeSH
- Drug Resistance, Neoplasm MeSH
- Antigens, Differentiation genetics MeSH
- Interferons pharmacology MeSH
- Humans MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Neoplastic Stem Cells drug effects metabolism radiation effects MeSH
- Neoplasm Proteins genetics MeSH
- Neoplasms drug therapy genetics radiotherapy MeSH
- Antineoplastic Agents pharmacology MeSH
- Gene Expression Regulation, Neoplastic drug effects radiation effects MeSH
- Up-Regulation drug effects radiation effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
Mitochondria and oxidative phosphorylation (OXPHOS) are emerging as intriguing targets for the efficient elimination of cancer cells. The specificity of this approach is aided by the capacity of non-proliferating non-cancerous cells to withstand oxidative insult induced by OXPHOS inhibition. Recently we discovered that mitochondrial targeting can also be employed to eliminate senescent cells, where it breaks the interplay between OXPHOS and ATP transporters that appear important for the maintenance of mitochondrial morphology and viability in the senescent setting. Hence, mitochondria/OXPHOS directed pharmacological interventions show promise in several clinically-relevant scenarios that call for selective removal of cancer and senescent cells.
- MeSH
- Adenosine Diphosphate metabolism MeSH
- Adenosine Triphosphate metabolism MeSH
- Biological Transport MeSH
- Cell Death MeSH
- Humans MeSH
- Mitochondria metabolism MeSH
- Neoplasms metabolism pathology MeSH
- Oxidative Phosphorylation MeSH
- Cell Proliferation MeSH
- Reactive Oxygen Species metabolism MeSH
- Cellular Senescence * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review 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 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
Many cancers arise at sites of infection and inflammation. Cellular senescence, a permanent state of cell cycle arrest that provides a barrier against tumorigenesis, is accompanied by elevated proinflammatory cytokines such as IL1, IL6, IL8 and TNFα. Here we demonstrate that media conditioned by cells undergoing any of the three main forms of senescence, i.e. replicative, oncogene- and drug-induced, contain high levels of IL1, IL6, and TGFb capable of inducing reactive oxygen species (ROS)-mediated DNA damage response (DDR). Persistent cytokine signaling and activated DDR evoke senescence in normal bystander cells, accompanied by activation of the JAK/STAT, TGFβ/SMAD and IL1/NFκB signaling pathways. Whereas inhibition of IL6/STAT signaling had no effect on DDR induction in bystander cells, inhibition of either TGFβ/SMAD or IL1/NFκB pathway resulted in decreased ROS production and reduced DDR in bystander cells. Simultaneous inhibition of both TGFβ/SMAD and IL1/NFκB pathways completely suppressed DDR indicating that IL1 and TGFβ cooperate to induce and/or maintain bystander senescence. Furthermore, the observed IL1- and TGFβ-induced expression of NAPDH oxidase Nox4 indicates a mechanistic link between the senescence-associated secretory phenotype (SASP) and DNA damage signaling as a feature shared by development of all major forms of paracrine bystander senescence.
- MeSH
- Cell Line MeSH
- Bystander Effect drug effects MeSH
- Etoposide pharmacology MeSH
- Genes, ras * MeSH
- Interleukin-1 metabolism MeSH
- Interleukin-6 metabolism MeSH
- Janus Kinases metabolism MeSH
- Culture Media, Conditioned metabolism MeSH
- Humans MeSH
- NADPH Oxidases metabolism MeSH
- NF-kappa B metabolism MeSH
- Oxidative Stress drug effects MeSH
- Paracrine Communication drug effects MeSH
- DNA Damage * MeSH
- Cell Proliferation * MeSH
- Smad Proteins metabolism MeSH
- RNA Interference MeSH
- Signal Transduction drug effects MeSH
- Cellular Senescence drug effects MeSH
- Transfection MeSH
- Transforming Growth Factor beta metabolism MeSH
- STAT Transcription Factors metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Tumor suppressor PML is induced under viral and genotoxic stresses by interferons and JAK-STAT signaling. However, the mechanism responsible for its cell type-specific regulation under non-stimulated conditions is poorly understood. To analyze the variation of PML expression, we utilized three human cell types, BJ fibroblasts and HeLa and U2OS cell lines, each with a distinct PML expression pattern. Analysis of JAK-STAT signaling in the three cell lines revealed differences in levels of activated STAT3 but not STAT1 correlating with PML mRNA and protein levels. RNAi-mediated knockdown of STAT3 decreased PML expression; both STAT3 level/activity and PML expression relied on IL6 secreted into culture media. We mapped the IL6-responsive sequence to an ISRE(-595/-628) element of the PML promoter. The PI3K/Akt/NFκB branch of IL6 signaling showed also cell-type dependence, being highest in BJ, intermediate in HeLa, and lowest in U2OS cells and correlated with IL6 secretion. RNAi-mediated knockdown of NEMO (NF-κ-B essential modulator), a key component of NFκB activation, suppressed NFκB targets LMP2 and IRF1 together with STAT3 and PML. Combined knockdown of STAT3 and NEMO did not further promote PML suppression, and it can be bypassed by exogenous IL6, indicating the NF-κB pathway acts upstream of JAK-STAT3 through induction of IL6. Our results indicate that the cell type-specific activity of IL6 signaling pathways governs PML expression under unperturbed growth conditions. As IL6 is induced in response to various viral and genotoxic stresses, this cytokine may regulate autocrine/paracrine induction of PML under these pathophysiological states as part of tissue adaptation to local stress.
- MeSH
- Leukemia, Promyelocytic, Acute metabolism pathology MeSH
- Chromatin Immunoprecipitation MeSH
- DNA Primers MeSH
- Fluorescent Antibody Technique, Indirect MeSH
- HeLa Cells MeSH
- Interleukin-6 metabolism MeSH
- Nuclear Proteins metabolism MeSH
- Janus Kinases metabolism MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Humans MeSH
- Tumor Suppressor Proteins metabolism MeSH
- Base Sequence MeSH
- Signal Transduction MeSH
- STAT Transcription Factors metabolism MeSH
- Transcription Factors metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
The Promyelocytic leukemia protein (PML) tumor suppressor is upregulated in several forms of cellular senescence, however the mechanism of its induction is elusive. Here we show that genotoxic drugs that induce senescence, such as 5-bromo-2'deoxyuridine (BrdU), thymidine (TMD), distamycin A (DMA), aphidicolin (APH), etoposide (ET) and camptothecin (CPT) all evoke expansion of PML nuclear compartment and its association with persistent DNA lesions in several human cancer cell lines and normal diploid fibroblasts. This phenomenon was accompanied by elevation of PML transcripts after treatment with BrdU, TMD, DMA and CPT. Chemical inhibition of all JAK kinases and RNAi-mediated knock-down of JAK1 suppressed PML expression, implicating JAK/STAT-mediated signaling in regulation of the PML gene. As PML protein stability remained unchanged after drug treatment, decreased protein turnover was unlikely to explain the senescence-associated increased abundance of PML. Furthermore, binding activity of Interferon Stimulated Response Element (ISRE) within the PML gene promoter, and suppression of reporter gene activity after deletion of ISRE from the PML promoter region suggested that drug-induced PML transcription is controlled via transcription factors interacting with this element. Collectively, our data show that upregulation of the PML tumor suppressor in cellular senescence triggered by diverse drugs including clinically used anti-cancer chemotherapeutics relies on stimulation of PML transcription by JAK/STAT-mediated signaling, possibly evoked by the autocrine/paracrine activities of senescence-associated cytokines.
- MeSH
- Cell Nucleus drug effects metabolism MeSH
- Transcription, Genetic drug effects MeSH
- Nuclear Proteins genetics metabolism MeSH
- Janus Kinase 1 metabolism MeSH
- Cell Compartmentation drug effects MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Proteins genetics metabolism MeSH
- Tumor Suppressor Protein p53 metabolism MeSH
- Neoplasms enzymology genetics pathology MeSH
- DNA Damage drug effects MeSH
- Antineoplastic Agents pharmacology MeSH
- Gene Expression Regulation, Leukemic drug effects MeSH
- Response Elements genetics MeSH
- Signal Transduction drug effects MeSH
- Cellular Senescence drug effects MeSH
- STAT Transcription Factors metabolism MeSH
- Transcription Factors genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
