Senescence and fibrosis in salivary gland aging and disease.

Salivary gland hypofunction is highly prevalent in aged and diseased individuals leading to significant discomfort and morbidity. One factor that contributes to salivary gland hypofunction is cellular aging, or senescence. Senescent cells can impair gland function by secreting paracrine-acting growth factors and cytokines, known as senescence-associated secretory phenotype (SASP) factors. These SASP factors stimulate inflammation, propagate the senescent phenotype through the bystander effect, and stimulate fibrosis. As senotherapeutics that target senescent cells have shown effectiveness in limiting disease manifestations in other conditions, there is interest in the use of these drugs to treat salivary gland hypofunction. In this review, we highlight the contribution of senescence and fibrosis to salivary gland pathologies. We also discuss therapeutic approaches to eliminate or modulate the senescent SASP phenotype for treating age-related salivary gland diseases and extending health span.

Arsenite toxicity is regulated by queuine availability and oxidation-induced reprogramming of the human tRNA epitranscriptome.

Cells respond to environmental stress by regulating gene expression at the level of both transcription and translation. The ∼50 modified ribonucleotides of the human epitranscriptome contribute to the latter, with mounting evidence that dynamic regulation of transfer RNA (tRNA) wobble modifications leads to selective translation of stress response proteins from codon-biased genes. Here we show that the response of human hepatocellular carcinoma cells to arsenite exposure is regulated by the availability of queuine, a micronutrient and essential precursor to the wobble modification queuosine (Q) on tRNAs reading GUN codons. Among oxidizing and alkylating agents at equitoxic concentrations, arsenite exposure caused an oxidant-specific increase in Q that correlated with up-regulation of proteins from codon-biased genes involved in energy metabolism. Limiting queuine increased arsenite-induced cell death, altered translation, increased reactive oxygen species levels, and caused mitochondrial dysfunction. In addition to demonstrating an epitranscriptomic facet of arsenite toxicity and response, our results highlight the links between environmental exposures, stress tolerance, RNA modifications, and micronutrients.

Selenoproteins and the senescence-associated epitranscriptome.

Selenium is a naturally found trace element, which provides multiple benefits including antioxidant, anticancer, and antiaging, as well as boosting immunity. One unique feature of selenium is its incorporation as selenocysteine, a rare 21st amino acid, into selenoproteins. Twenty-five human selenoproteins have been discovered, and a majority of these serve as crucial antioxidant enzymes for redox homeostasis. Unlike other amino acids, incorporation of selenocysteine requires a distinctive UGA stop codon recoding mechanism. Although many studies correlating selenium, selenoproteins, aging, and senescence have been performed, it has not yet been explored if the upstream events regulating selenoprotein synthesis play a role in senescence-associated pathologies. The epitranscriptomic writer alkylation repair homolog 8 (ALKBH8) is critical for selenoprotein production, and its deficiency can significantly decrease levels of selenoproteins that are essential for reactive oxygen species (ROS) detoxification, and increase oxidative stress, one of the major drivers of cellular senescence. Here, we review the potential role of epitranscriptomic marks that govern selenocysteine utilization in regulating the senescence program.

Boronate probe-based hydrogen peroxide detection with AlGaN/GaN HEMT sensor.

The results from this study demonstrate the potential of an AlGaN/GaN high electron mobility transistor sensor for the detection of reactive and transient biological molecules such as hydrogen peroxide. A boronate-based fluorescent probe was used with this device to detect the presence of micromolar levels of hydrogen peroxide typically associated with intracellular processes. The real-time electrical response of the high electron mobility transistor sensor showed a gradual decrease in the two-dimensional electron gas current as the reaction proceeded over time. A corresponding increase in the emission intensity was measured from the fluorescent probe with the progression of the reaction. The fluorescence from the boronate probe was used as an indicator to confirm the detection of hydrogen peroxide. These results demonstrate the dynamic measurement capability of AlGaN/GaN high electron mobility transistor sensors in monitoring real-time reactions of reactive oxygen species such as hydrogen peroxide.

Redox and mTOR-dependent regulation of plasma lamellar calcium influx controls the senescence-associated secretory phenotype.

IMPACT STATEMENT: Through its ability to evoke responses from cells in a paracrine fashion, the senescence-associated secretory phenotype (SASP) has been linked to numerous age-associated disease pathologies including tumor invasion, cardiovascular dysfunction, neuroinflammation, osteoarthritis, and renal disease. Strategies which limit the amplitude and duration of SASP serve to delay age-related degenerative decline. Here we demonstrate that the SASP regulation is linked to shifts in intracellular Ca2+ homeostasis and strategies which rescue redox-dependent calcium entry including enzymatic H2O2 scavenging, TRP modulation, or mTOR inhibition block SASP and TRPC6 gene expression. As Ca2+ is indispensable for secretion from both secretory and non-secretory cells, it is exciting to speculate that the expression of plasma lamellar TRP channels critical for the maintenance of intracellular Ca2+ homeostasis may be coordinately regulated with the SASP.

The epitranscriptomic writer ALKBH8 drives tolerance and protects mouse lungs from the environmental pollutant naphthalene.

The epitranscriptomic writer Alkylation Repair Homolog 8 (ALKBH8) is a transfer RNA (tRNA) methyltransferase that modifies the wobble uridine of selenocysteine tRNA to promote the specialized translation of selenoproteins. Using Alkbh8 deficient (Alkbh8def) mice, we have investigated the importance of epitranscriptomic systems in the response to naphthalene, an abundant polycyclic aromatic hydrocarbon and environmental toxicant. We performed basal lung analysis and naphthalene exposure studies using wild type (WT), Alkbh8def and Cyp2abfgs-null mice, the latter of which lack the cytochrome P450 enzymes required for naphthalene bioactivation. Under basal conditions, lungs from Alkbh8def mice have increased markers of oxidative stress and decreased thioredoxin reductase protein levels, and have reprogrammed gene expression to differentially regulate stress response transcripts. Alkbh8def mice are more sensitive to naphthalene induced death than WT, showing higher susceptibility to lung damage at the cellular and molecular levels. Further, WT mice develop a tolerance to naphthalene after 3 days, defined as resistance to a high challenging dose after repeated exposures, which is absent in Alkbh8def mice. We conclude that the epitranscriptomic writer ALKBH8 plays a protective role against naphthalene-induced lung dysfunction and promotes naphthalene tolerance. Our work provides an early example of how epitranscriptomic systems can regulate the response to environmental stress in vivo.

Loss of epitranscriptomic control of selenocysteine utilization engages senescence and mitochondrial reprogramming☆.

Critically important to the maintenance of the glutathione (GSH) redox cycle are the activities of many selenocysteine-containing GSH metabolizing enzymes whose translation is controlled by the epitranscriptomic writer alkylation repair homolog 8 (ALKBH8). ALKBH8 is a tRNA methyltransferase that methylates the wobble uridine of specific tRNAs to regulate the synthesis of selenoproteins. Here we demonstrate that a deficiency in the writer ALKBH8 (Alkbh8def), alters selenoprotein levels and engages senescence, regulates stress response genes and promotes mitochondrial reprogramming. Alkbh8def mouse embryonic fibroblasts (MEFs) increase many hallmarks of senescence, including senescence associated ß-galactosidase, heterocromatic foci, the cyclin dependent kinase inhibitor p16Ink4a, markers of mitochondrial dynamics as well as the senescence associated secretory phenotype (SASP). Alkbh8def cells also acquire a stress resistance phenotype that is accompanied by an increase in a number redox-modifying transcripts. In addition, Alkbh8def MEFs undergo a metabolic shift that is highlighted by a striking increase in the level of uncoupling protein 2 (UCP2) which enhances oxygen consumption and promotes a reliance on glycolytic metabolism. Finally, we have shown that the Alkbh8 deficiency can be exploited and corresponding MEFs are killed by glycolytic inhibition. Our work demonstrates that defects in an epitransciptomic writer promote senescence and mitochondrial reprogramming and unveils a novel adaptive mechanism for coping with defects in selenocysteine utilization.

Epitranscriptomic systems regulate the translation of reactive oxygen species detoxifying and disease linked selenoproteins.

Here we highlight the role of epitranscriptomic systems in post-transcriptional regulation, with a specific focus on RNA modifying writers required for the incorporation of the 21st amino acid selenocysteine during translation, and the pathologies linked to epitranscriptomic and selenoprotein defects. Epitranscriptomic marks in the form of enzyme-catalyzed modifications to RNA have been shown to be important signals regulating translation, with defects linked to altered development, intellectual impairment, and cancer. Modifications to rRNA, mRNA and tRNA can affect their structure and function, while the levels of these dynamic tRNA-specific epitranscriptomic marks are stress-regulated to control translation. The tRNA for selenocysteine contains five distinct epitranscriptomic marks and the ALKBH8 writer for the wobble uridine (U) has been shown to be vital for the translation of the glutathione peroxidase (GPX) and thioredoxin reductase (TRXR) family of selenoproteins. The reactive oxygen species (ROS) detoxifying selenocysteine containing proteins are a prime examples of how specialized translation can be regulated by specific tRNA modifications working in conjunction with distinct codon usage patterns, RNA binding proteins and specific 3' untranslated region (UTR) signals. We highlight the important role of selenoproteins in detoxifying ROS and provide details on how epitranscriptomic marks and selenoproteins can play key roles in and maintaining mitochondrial function and preventing disease.

A Dynamic Culture Method to Produce Ovarian Cancer Spheroids under Physiologically-Relevant Shear Stress.

The transcoelomic metastasis pathway is an alternative to traditional lymphatic/hematogenic metastasis. It is most frequently observed in ovarian cancer, though it has been documented in colon and gastric cancers as well. In transcoelomic metastasis, primary tumor cells are released into the abdominal cavity and form cell aggregates known as spheroids. These spheroids travel through the peritoneal fluid and implant at secondary sites, leading to the formation of new tumor lesions in the peritoneal lining and the organs in the cavity. Models of this process that incorporate the fluid shear stress (FSS) experienced by these spheroids are few, and most have not been fully characterized. Proposed herein is the adaption of a known dynamic cell culture system, the orbital shaker, to create an environment with physiologically-relevant FSS for spheroid formation. Experimental conditions (rotation speed, well size and cell density) were optimized to achieve physiologically-relevant FSS while facilitating the formation of spheroids that are also of a physiologically-relevant size. The FSS improves the roundness and size consistency of spheroids versus equivalent static methods and are even comparable to established high-throughput arrays, while maintaining nearly equivalent viability. This effect was seen in both highly metastatic and modestly metastatic cell lines. The spheroids generated using this technique were fully amenable to functional assays and will allow for better characterization of FSS's effects on metastatic behavior and serve as a drug screening platform. This model can also be built upon in the future by adding more aspects of the peritoneal microenvironment, further enhancing its in vivo relevance.

Senescence in chronic allograft nephropathy.

Despite increasing numbers of patients on dialysis, the numbers of renal transplants performed yearly have remained relatively static. During the last 50 years, there have been many advances in the pharmacology of prevention of organ rejection. However, most patients will suffer from a slow but steady decline in renal function leading to graft loss. The most common cause of long-term graft loss is chronic allograft nephropathy (CAN). Therefore, elucidating and understanding the mechanisms involved in CAN is crucial for achieving better posttransplant outcomes. It is thought that the development of epithelial to mesenchymal transition (EMT) in proximal tubules is one of the first steps towards CAN, and has been shown to be a result of cellular senescence. Cells undergoing senescence acquire a senescence associated secretory phenotype (SASP) leading to the production of interleukin-1 alpha (IL-1α), which has been implicated in several degenerative and inflammatory processes including renal disease. A central mediator in SASP activation is the production of reactive oxygen species (ROS), which are produced in response to numerous physiological and pathological stimuli. This review explores the connection between SASP and the development of EMT/CAN in an effort to suggest future directions for research leading to improved long-term graft outcomes.

Mitochondrial ROS control of cancer.

Mitochondria serves a primary role in energy maintenance but also function to govern levels of mitochondria-derived reactive oxygen species (mROS). ROS have long been established to play a critical role in tumorigenesis and are now considered to be integral to the regulation of diverse signaling networks that drive proliferation, tumor cell survival and malignant progression. mROS can damage DNA, activate oncogenes, block the function of tumor suppressors and drive migratory signaling. The mitochondrion's oxidant scavenging systems including SOD2, Grx2, GPrx, Trx and TrxR are key of the cellular redox tone. These mitochondrial antioxidant systems serve to tightly control the levels of the primary ROS signaling species, H2O2. The coordinated control of mROS levels is also coupled to the activity of the primary H2O2 consuming enzymes of the mitochondria which are reliant on the epitranscriptomic control of selenocysteine incorporation. This review highlights the interplay between these many oncogenic signaling networks, mROS and the H2O2 emitting and consuming capacity of the mitochondria.

Redox control of senescence and age-related disease.

The signaling networks that drive the aging process, associated functional deterioration, and pathologies has captured the scientific community's attention for decades. While many theories exist to explain the aging process, the production of reactive oxygen species (ROS) provides a signaling link between engagement of cellular senescence and several age-associated pathologies. Cellular senescence has evolved to restrict tumor progression but the accompanying senescence-associated secretory phenotype (SASP) promotes pathogenic pathways. Here, we review known biological theories of aging and how ROS mechanistically control senescence and the aging process. We also describe the redox-regulated signaling networks controlling the SASP and its important role in driving age-related diseases. Finally, we discuss progress in designing therapeutic strategies that manipulate the cellular redox environment to restrict age-associated pathology.

Featured Article: Nanoenhanced matrix metalloproteinase-responsive delivery vehicles for disease resolution and imaging.

The wide array of proteases, including matrix metalloproteinases, produced in response to many pathogenic insults, confers a unique proteolytic signature which is often disease specific and provides a potential therapeutic target for drug delivery. Here we propose the use of collagen-based nanoenhanced matrix metalloproteinase-responsive delivery vehicles that display matrix metalloproteinase-specific degradation in diverse in vitro models of proteolysis. We demonstrate that collagen particles comprised of protease substrates (primarily collagen) can be made of uniform size and loaded efficiently with assorted cargo including fluorescently labeled mesoporous silica, magnetic nanoparticles, proteins and antioxidants. We also demonstrate that pathologic concentrations of proteases produced in situ or in vitro display protease-specific cargo release. Additionally, we show that the collagen-based particles display bright fluorescence when loaded with a fluorophore, and have the potential to be used as vehicles for targeted delivery of drugs or imaging agents to regions of high proteolytic activity.

Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function.

Comparative analysis of redox and inflammatory properties of pristine nanomaterials and commonly used semiconductor manufacturing nano-abrasives.

Continued expansion of the nanotechnology industry has necessitated the self-assessment of manufacturing processes, specifically in regards to understanding the health related aspects following exposure to nanomaterials. There exists a growing concern over potential occupational exposure in the semiconductor industry where Al2O3, CeO2 and SiO2 nanoparticles are commonly featured as part of the chemical mechanical planarization (CMP) process. Chronic exposure to toxicants can result not only in acute cytotoxicity but also initiation of a chronic inflammatory state associated with diverse pathologies. In the current investigation, pristine nanoparticles and CMP slurry formulations of Al2O3, SiO2 and CeO2 were employed to assess their ability to induce cytotoxicity, inflammatory responses and reactive oxygen species in a mouse alveolar macrophage cell model. The pristine nanoparticles and slurries were not intrinsically cytotoxic and did not generate free radicals but were found to act as scavengers in the presence of an oxidant stimulant. Al2O3 and SiO2 nanoparticles increased levels of pro-inflammatory cytokines while pristine SiO2 nanoparticles induced generation of F2-Isoprostanes. In co-treatment studies, the pristine nanomaterials modulated the response to the inflammatory stimulant lipopolysaccharide. The studies have established that pristine nanoparticles and slurries do not impact the cells in a similar way indicating that they should not be used as slurry substitutes in toxicity evaluations. Further, we have defined how an alveolar cell line, which would likely be the first challenged upon nanomaterial aerosolization, responds to diverse mixtures of nanomaterials. Moreover, our findings reinforce the importance of using multiple analytic methods to define the redox state of the cell following exposure to commonly used industrial nanomaterials and toxicants.

Alkbh8 Regulates Selenocysteine-Protein Expression to Protect against Reactive Oxygen Species Damage.

Environmental and metabolic sources of reactive oxygen species (ROS) can damage DNA, proteins and lipids to promote disease. Regulation of gene expression can prevent this damage and can include increased transcription, translation and post translational modification. Cellular responses to ROS play important roles in disease prevention, with deficiencies linked to cancer, neurodegeneration and ageing. Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8. Using a new gene targeted knockout mouse cell system, we show that Alkbh8-/- embryonic fibroblasts (MEFs) display elevated ROS levels, increased DNA and lipid damage and hallmarks of cellular stress. We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families. We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS. These studies define Alkbh8 and tRNA modifications as central regulators of cellular oxidative stress responses in mammalian systems. In addition they highlight a new animal model for use in environmental and cancer studies and link translational regulation to the prevention of DNA and lipid damage.

Redox-sensitive gene-regulatory events controlling aberrant matrix metalloproteinase-1 expression.

Aberrant matrix metalloproteinase-1 (MMP-1) expression contributes to the pathogenesis of many degenerative disease processes that are associated with increased oxidative damage or stress. We and others have established that shifts in steady-state H2O2 production resulting from enforced antioxidant gene expression, senescence, or UV irradiation control MMP-1 expression. Here we establish that histone deacetylase-2 (HDAC2) protein levels and its occupancy of the MMP-1 promoter are decreased in response to enforced manganese superoxide dismutase (Sod2) expression. Inhibition of HDAC activity further accentuates the redox-dependent expression of MMP-1. Sod2-dependent decreases in HDAC2 are associated with increases in a proteasome-sensitive pool of ubiquitinylated HDAC2 and MMP-1-specific histone H3 acetylation. Sod2 overexpression also enhanced recruitment of Ets-1, c-Jun, c-Fos, and the histone acetyltransferase PCAF to the distal and proximal regions of the MMP-1 promoter. Furthermore, the Sod2-dependent expression of MMP-1 can be reversed by silencing the transcriptional activator c-Jun. All of the above Sod2-dependent alterations are largely reversed by catalase coexpression, indicating that the redox control of MMP-1 is H2O2-dependent. These findings identify a novel redox regulation of MMP-1 transcription that involves site-specific promoter recruitment of both activating factors and chromatin-modifying enzymes, which converge to maximally drive MMP-1 gene expression.

Micropatterning of 3D Microenvironments for Living Biosensor Applications.

Micro-scale printing and patterning of living cells has multiple applications including tissue engineering, cell signaling assays, and the fabrication of cell-based biosensors. In this work, a molecular printing instrument, the Bioforce Nano eNabler, was modified to enable micron-scale -quill-pen based printing of mammalian cells in a 3D hyaluronan/gelatin based hydrogel. Specifically, photo-initiated -thiol-ene click chemistry was used to couple the thiol groups of thiolated hyaluronan/thiolated gelatin to the alkene groups of 4-arm polyethylene glycol (PEG)-norbornene molecules. Rapid photopolymerization enabled direct printing and controlled curing of living cells within the hydrogel matrix. The resulting hydrogels were biocompatible with human adipose-derived stem cells, NIH-3T3 cells, and mouse embryonic stem cells. The utility of this printing approach was also explored for cell-based biosensors. Micro-printed cells expressing a redox sensitive variant of the green fluorescent protein (roGFP-R12) showed a measurable fluorescent response to addition of oxidizing and then reducing agents. This work represents a novel approach to micron-scale cell patterning, and its potential for living, cell-based biosensors.

Intracellular redox status controls membrane localization of pro- and anti-migratory signaling molecules.

Shifts in intracellular Reactive Oxygen Species (ROS) have been shown to contribute to carcinogenesis and to tumor progression. In addition to DNA and cell damage by surges in ROS, sub-lethal increases in ROS are implicated in regulating cellular signaling that enhances pro-metastatic behavior. We previously showed that subtle increases in endogenous H2O2 regulate migratory and invasive behavior of metastatic bladder cancer cells through phosphatase inhibition and consequential phosphorylation of p130cas, an adapter of the FAK signaling pathway. We further showed that enhanced redox status contributed to enhanced localization of p130cas to the membrane of metastatic cells. Here we show that this signaling complex can similarly be induced in a redox-engineered cell culture model that enables regulation of intracellular steady state H2O2 level by enforced expression of superoxide dismutase 2 (Sod2) and catalase. Expression of Sod2 leads to enhanced p130cas phosphorylation in HT-1080 fibrosarcoma and UM-UC-6 bladder cancer cells. These changes are mediated by H2O2, as co-expression of Catalase abrogates p130cas phosphorylation and its interaction with the adapter protein Crk. Importantly, we establish that the redox environment influence the localization of the tumor suppressor and phosphatase PTEN, in both redox-engineered and metastatic bladder cancer cells that display endogenous increases in H2O2. Importantly, PTEN oxidation leads to its dissociation from the plasma membrane. This indicates that oxidation of PTEN not only influences its activity, but also regulates its cellular localization, effectively removing it from its primary site of lipid phosphatase activity. These data introduce hitherto unappreciated paradigms whereby ROS can reciprocally regulate the cellular localization of pro- and anti-migratory signaling molecules, p130cas and PTEN, respectively. These data further confirm that altering antioxidant status and the intracellular ROS environment can have profound effects on pro-metastatic signaling pathways.

Redox control of the senescence regulator interleukin-1α and the secretory phenotype.

Senescent cells accumulate in aged tissue and are causally linked to age-associated tissue degeneration. These non-dividing, metabolically active cells are highly secretory and alter tissue homeostasis, creating an environment conducive to metastatic disease progression. IL-1α is a key senescence-associated (SA) proinflammatory cytokine that acts as a critical upstream regulator of the SA secretory phenotype (SASP). We established that SA shifts in steady-state H2O2 and intracellular Ca(2+) levels caused an increase in IL-1α expression and processing. The increase in intracellular Ca(2+) promoted calpain activation and increased the proteolytic cleavage of IL-1α. Antioxidants and low oxygen tension prevented SA IL-1α expression and restricted expression of SASP components IL-6 and IL-8. Ca(2+) chelation or calpain inhibition prevented SA processing of IL-1α and its ability to induce downstream cytokine expression. Conditioned medium from senescent cells treated with antioxidants or Ca(2+) chelators or cultured in low oxygen markedly reduced the invasive capacity of proximal metastatic cancer cells. In this paracrine fashion, senescent cells promoted invasion by inducing an epithelial-mesenchymal transition, actin reorganization, and cellular polarization of neighboring cancer cells. Collectively, these findings demonstrate how SA alterations in the redox state and Ca(2+) homeostasis modulate the inflammatory phenotype through the regulation of the SASP initiator IL-1α, creating a microenvironment permissive to tumor invasion.