Mitochondrial Quality Control via the PGC1α-TFEB Signaling Pathway Is Compromised by Parkin Q311X Mutation But Independently Restored by Rapamycin.

Following its activation by PINK1, parkin is recruited to depolarized mitochondria where it ubiquitinates outer mitochondrial membrane proteins, initiating lysosomal-mediated degradation of these organelles. Mutations in the gene encoding parkin, PARK2, result in both familial and sporadic forms of Parkinson's disease (PD) in conjunction with reductions in removal of damaged mitochondria. In contrast to what has been reported for other PARK2 mutations, expression of the Q311X mutation in vivo in mice appears to involve a downstream step in the autophagic pathway at the level of lysosomal function. This coincides with increased PARIS expression and reduced expression of a reciprocal signaling pathway involving the master mitochondrial regulator peroxisome proliferator-activated receptor-gamma coactivator (PGC1α) and the lysosomal regulator transcription factor EB (TFEB). Treatment with rapamycin was found to independently restore PGC1α-TFEB signaling in a manner not requiring parkin activity and to abrogate impairment of mitochondrial quality control and neurodegenerative features associated with this in vivo model. Losses in PGC1α-TFEB signaling in cultured rat DAergic cells expressing the Q311X mutation associated with reduced mitochondrial function and cell viability were found to be PARIS-dependent and to be independently restored by rapamycin in a manner requiring TFEB. Studies in human iPSC-derived neurons demonstrate that TFEB induction can restore mitochondrial function and cell viability in a mitochondrially compromised human cell model. Based on these data, we propose that the parkin Q311X mutation impacts on mitochondrial quality control via PARIS-mediated regulation of PGC1α-TFEB signaling and that this can be independently restored via upregulation of TFEB function. SIGNIFICANCE STATEMENT: Mutations in PARK2 are generally associated with loss in ability to interact with PINK1, impacting on autophagic initiation. Our data suggest that, in the case of at least one parkin mutation, Q311X, detrimental effects are due to inhibition at the level of downstream lysosomal function. Mechanistically, this involves elevations in PARIS protein levels and subsequent effects on PGC1α-TFEB signaling that normally regulates mitochondrial quality control. Treatment with rapamycin independently restores PGC1α-TFEB signaling in a manner not requiring parkin activity and abrogates subsequent mitochondrial impairment and neuronal cell loss. Taken in total, our data suggest that the parkin Q311X mutation impacts on mitochondrial quality control via PARIS-mediated regulation of PGC1α-TFEB signaling and that this can be independently restored via rapamycin.

Amyloid ß accelerates age-related proteome-wide protein insolubility.

Loss of proteostasis is a highly conserved feature of aging across model organisms and results in the accumulation of insoluble protein aggregates. Protein insolubility is also a unifying feature of major age-related neurodegenerative diseases, including Alzheimer's Disease (AD), in which hundreds of insoluble proteins associate with aggregated amyloid beta (Aß) in senile plaques. Despite the connection between aging and AD risk, therapeutic approaches to date have overlooked aging-driven generalized protein insolubility as a contributing factor. However, proteins that become insoluble during aging in model organisms are capable of accelerating Aß aggregation in vitro and lifespan in vivo. Here, using an unbiased proteomics approach, we questioned the relationship between Aß and age-related protein insolubility. Specifically, we uncovered that Aß expression drives proteome-wide protein insolubility in C. elegans, even in young animals, and this insoluble proteome is highly similar to the insoluble proteome driven by normal aging, this vulnerable sub-proteome we term the core insoluble proteome (CIP). We show that the CIP is enriched with proteins that modify Aß toxicity in vivo, suggesting the possibility of a vicious feedforward cycle in the context of AD. Importantly, using human genome-wide association studies (GWAS), we show that the CIP is replete with biological processes implicated not only in neurodegenerative diseases but also across a broad array of chronic, age-related diseases (CARDs). This provides suggestive evidence that age-related loss of proteostasis could play a role in general CARD risk. Finally, we show that the geroprotective, gut-derived metabolite, Urolithin A, relieves Aß toxicity, supporting its use in clinical trials for dementia and age-related diseases.

Antioxidants green tea extract and nordihydroguaiaretic acid confer species and strain-specific lifespan and health effects in Caenorhabditis nematodes.

The Caenorhabditis Intervention Testing Program (CITP) is an NIH-funded research consortium of investigators who conduct analyses at three independent sites to identify chemical interventions that reproducibly promote health and lifespan in a robust manner. The founding principle of the CITP is that compounds with positive effects across a genetically diverse panel of Caenorhabditis species and strains are likely engaging conserved biochemical pathways to exert their effects. As such, interventions that are broadly efficacious might be considered prominent compounds for translation for pre-clinical research and human clinical applications. Here, we report results generated using a recently streamlined pipeline approach for the evaluation of the effects of chemical compounds on lifespan and health. We studied five compounds previously shown to extend C. elegans lifespan or thought to promote mammalian health: 17α-estradiol, acarbose, green tea extract, nordihydroguaiaretic acid, and rapamycin. We found that green tea extract and nordihydroguaiaretic acid extend Caenorhabditis lifespan in a species-specific manner. Additionally, these two antioxidants conferred assay-specific effects in some studies-for example, decreasing survival for certain genetic backgrounds in manual survival assays in contrast with extended lifespan as assayed using automated C. elegans Lifespan Machines. We also observed that GTE and NDGA impact on older adult mobility capacity is dependent on genetic background, and that GTE reduces oxidative stress resistance in some Caenorhabditis strains. Overall, our analysis of the five compounds supports the general idea that genetic background and assay type can influence lifespan and health effects of compounds, and underscores that lifespan and health can be uncoupled by chemical interventions.

DNA-SCARS: distinct nuclear structures that sustain damage-induced senescence growth arrest and inflammatory cytokine secretion.

DNA damage can induce a tumor suppressive response termed cellular senescence. Damaged senescent cells permanently arrest growth, secrete inflammatory cytokines and other proteins and harbor persistent nuclear foci that contain DNA damage response (DDR) proteins. To understand how persistent damage foci differ from transient foci that mark repairable DNA lesions, we identify sequential events that differentiate transient foci from persistent foci, which we term 'DNA segments with chromatin alterations reinforcing senescence' (DNA-SCARS). Unlike transient foci, DNA-SCARS associate with PML nuclear bodies, lack the DNA repair proteins RPA and RAD51, lack single-stranded DNA and DNA synthesis and accumulate activated forms of the DDR mediators CHK2 and p53. DNA-SCARS form independently of p53, pRB and several other checkpoint and repair proteins but require p53 and pRb to trigger the senescence growth arrest. Importantly, depletion of the DNA-SCARS-stabilizing component histone H2AX did not deplete 53BP1 from DNA-SCARS but diminished the presence of MDC1 and activated CHK2. Furthermore, depletion of H2AX reduced both the p53-dependent senescence growth arrest and p53-independent cytokine secretion. DNA-SCARS were also observed following severe damage to multiple human cell types and mouse tissues, suggesting that they can be used in combination with other markers to identify senescent cells. Thus, DNA-SCARS are dynamically formed distinct structures that functionally regulate multiple aspects of the senescent phenotype.

Amyloid ß accelerates age-related proteome-wide protein insolubility.

Loss of proteostasis is a highly conserved feature of aging across model organisms and typically results in the accumulation of insoluble protein aggregates. Protein insolubility is a central feature of major age-related neurodegenerative diseases, including Alzheimer's Disease (AD), where hundreds of insoluble proteins associate with aggregated amyloid beta (Aß) in senile plaques. Moreover, proteins that become insoluble during aging in model organisms are capable of accelerating Aß aggregation in vitro. Despite the connection between aging and AD risk, therapeutic approaches to date have overlooked aging-driven protein insolubility as a contributory factor. Here, using an unbiased proteomics approach, we questioned the relationship between Aß and age-related protein insolubility. We demonstrate that Aß expression drives proteome-wide protein insolubility in C. elegans and this insoluble proteome closely resembles the insoluble proteome driven by normal aging, suggesting the possibility of a vicious feedforward cycle of aggregation in the context of AD. Importantly, using human genome-wide association studies (GWAS), we show that the CIP is replete with biological processes implicated not only in neurodegenerative diseases but also across a broad array of chronic, age-related diseases (CARDs). This provides suggestive evidence that age-related loss of proteostasis could play a role in general CARD risk. Finally, we show that the CIP is enriched with proteins that modulate the toxic effects of Aß and that the gut-derived metabolite, Urolithin A, relieves Aß toxicity, supporting its use in clinical trials for dementia and other age-related diseases.

Simultaneous neuronal expression of human amyloid-ß and Tau genes drives global phenotypic and multi-omic changes in C. elegans.

Alzheimer's disease and Alzheimer's related diseases (ADRD) are a class of prevalent age-related neurodegenerative disorders characterized by the accumulation of amyloid- ß (Aß) plaques and Tau neurofibrillary tangles. The intricate interplay between Aß and Tau proteins requires further investigation to better understand the precise mechanisms underlying disease pathology. The nematode Caenorhabditis elegans ( C. elegans ) serves as an invaluable model organism for studying aging and neurodegenerative diseases. Here we performed an unbiased systems analysis of a C. elegans strain expressing both Aß and Tau proteins within neurons. Intriguingly, even at an early stage of adulthood, we observed reproductive impairments and mitochondrial dysfunction consistent with substantial disruptions in mRNA transcript abundance, protein solubility, and metabolite levels. Notably, the simultaneous expression of these two neurotoxic proteins exhibited a synergistic effect, leading to accelerated aging in the model organism. Our comprehensive findings shed new light on the intricate relationship between normal aging processes and the etiology of ADRD. Specifically, we demonstrate the alterations to metabolic functions precede age-related neurotoxicity, offering critical insights into potential therapeutic strategies.

Cell surface-bound IL-1alpha is an upstream regulator of the senescence-associated IL-6/IL-8 cytokine network.

Inflammation underlies most age-related diseases, including cancer, but the etiology is poorly understood. One proposed factor is the presence of senescent cells, which increase with age. The senescence response arrests the proliferation of potentially oncogenic cells, and most senescent cells secrete high levels of proinflammatory cytokines and other proteins. The complex senescence-associated secretory phenotype is likely regulated at multiple levels, most of which are unknown. We show that cell surface-bound IL-1alpha is essential for signaling the senescence-associated secretion of IL-6 and IL-8, 2 proinflammatory cytokines that also reinforce the senescence growth arrest. Senescent human fibroblasts expressed high levels of IL-1alpha mRNA, intracellular protein, and cell surface-associated protein, but secreted very little protein. An IL-1 receptor (IL1R) antagonist, neutralizing IL-1alpha antibodies, and IL-1alpha depletion by RNA interference all markedly reduced senescence-associated IL-6/IL-8 secretion. Depletion of the key IL-1R signaling component IRAK1 also suppressed this secretion, and IL-1alpha neutralizing antibodies prevented IRAK1 degradation, indicating engagement of the IL-1R signaling pathway. Furthermore, IL-1alpha depletion reduced the DNA binding activity of NF-kappaB and C/EBPbeta, which stimulate IL-6/IL-8 transcription. IL-1alpha was a general regulator of senescence-associated IL-6/IL-8 secretion because IL-1alpha blockade reduced IL-6/IL-8 secretion whether cells senesced owing to DNA damage, replicative exhaustion, oncogenic RAS, or chromatin relaxation. Furthermore, conditioned medium from IL-1alpha-depleted senescent cells markedly reduced the IL-6/IL-8-dependent invasiveness of metastatic cancer cells, indicating that IL-1alpha regulates the biological effects of these cytokines. Thus, cell surface IL-1alpha is an essential cell-autonomous regulator of the senescence-associated IL-6/IL-8 cytokine network.

An aco-2::gfp knock-in enables the monitoring of mitochondrial morphology throughout C. elegans lifespan.

We used CRISPR/Cas9 gene editing in C. elegans in order to fluorescently tag endogenous aconitase-2 (ACO-2). ACO-2 is a mitochondrially localized protein, and the aco-2::gfp strain enabled the examination of native mitochondrial morphology in live animals. Here we validate that the aco-2::gfp strain displays the prototypic changes in mitochondrial morphology known to occur during aging and upon paraquat (PQ) induced mitochondrial stress. We also provide evidence that the ACO-2::GFP reporter can serve as a superior means for tracking mitochondrial morphology than conventional MitoTracker dyes-especially in aged-worms.

Metformin treatment of diverse Caenorhabditis species reveals the importance of genetic background in longevity and healthspan extension outcomes.

Metformin, the most commonly prescribed anti-diabetes medication, has multiple reported health benefits, including lowering the risks of cardiovascular disease and cancer, improving cognitive function with age, extending survival in diabetic patients, and, in several animal models, promoting youthful physiology and lifespan. Due to its longevity and health effects, metformin is now the focus of the first proposed clinical trial of an anti-aging drug-the Targeting Aging with Metformin (TAME) program. Genetic variation will likely influence outcomes when studying metformin health effects in human populations. To test for metformin impact in diverse genetic backgrounds, we measured lifespan and healthspan effects of metformin treatment in three Caenorhabditis species representing genetic variability greater than that between mice and humans. We show that metformin increases median survival in three C. elegans strains, but not in C. briggsae and C. tropicalis strains. In C. briggsae, metformin either has no impact on survival or decreases lifespan. In C. tropicalis, metformin decreases median survival in a dose-dependent manner. We show that metformin prolongs the period of youthful vigor in all C. elegans strains and in two C. briggsae strains, but that metformin has a negative impact on the locomotion of C. tropicalis strains. Our data demonstrate that metformin can be a robust promoter of healthy aging across different genetic backgrounds, but that genetic variation can determine whether metformin has positive, neutral, or negative lifespan/healthspan impact. These results underscore the importance of tailoring treatment to individuals when testing for metformin health benefits in diverse human populations.

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow.

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble proteins over 40% of the time results in an extension of the lifespan in C. elegans, suggesting that many of these proteins are key determinants of the aging process. Isolation and quantitative identification of these insoluble proteins are crucial to understand key biological processes that occur during aging. Here, we present a modified and improved protocol that details how to extract and isolate the SDS-insoluble proteins (insolublome) from C. elegans more efficiently to streamline mass spectrometric workflows via a novel label-free quantitative proteomics analysis. This improved protocol utilizes a highly efficient sonicator for worm lysis that greatly increases efficiency for protein extraction and allows us to use significantly less starting material (approximately 3,000 worms) than in previous protocols (typically using at least 40,000 worms). Subsequent quantitative proteomic analysis of the insolublome was performed using data-dependent acquisition (DDA) for protein discovery and identification and data-independent acquisition (DIA) for comprehensive and more accurate protein quantification. Bioinformatic analysis of quantified proteins provides potential candidates that can be easily followed up with other molecular methods in C. elegans. With this workflow, we routinely identify more than 1000 proteins and quantify more than 500 proteins. This new protocol enables efficient compound screening with C. elegans. Here, we validated and applied this improved protocol to wild-type C. elegans N2-Bristol strain and confirmed that aged day-10 N2 worms showed greater accumulation of the insolublome than day-2 young worms.

Alpha-Ketoglutarate, an Endogenous Metabolite, Extends Lifespan and Compresses Morbidity in Aging Mice.

Metabolism and aging are tightly connected. Alpha-ketoglutarate is a key metabolite in the tricarboxylic acid (TCA) cycle, and its levels change upon fasting, exercise, and aging. Here, we investigate the effect of alpha-ketoglutarate (delivered in the form of a calcium salt, CaAKG) on healthspan and lifespan in C57BL/6 mice. To probe the relationship between healthspan and lifespan extension in mammals, we performed a series of longitudinal, clinically relevant measurements. We find that CaAKG promotes a longer, healthier life associated with a decrease in levels of systemic inflammatory cytokines. We propose that induction of IL-10 by dietary AKG suppresses chronic inflammation, leading to health benefits. By simultaneously reducing frailty and enhancing longevity, AKG, at least in the murine model, results in a compression of morbidity.

Two faces of p53: aging and tumor suppression.

The p53 tumor suppressor protein, often termed guardian of the genome, integrates diverse physiological signals in mammalian cells. In response to stress signals, perhaps the best studied of which is the response to DNA damage, p53 becomes functionally active and triggers either a transient cell cycle arrest, cell death (apoptosis) or permanent cell cycle arrest (cellular senescence). Both apoptosis and cellular senescence are potent tumor suppressor mechanisms that irreversibly prevent damaged cells from undergoing neoplastic transformation. However, both processes can also deplete renewable tissues of proliferation-competent progenitor or stem cells. Such depletion, in turn, can compromise the structure and function of tissues, which is a hallmark of aging. Moreover, whereas apoptotic cells are by definition eliminated from tissues, senescent cells can persist, acquire altered functions, and thus alter tissue microenvironments in ways that can promote both cancer and aging phenotypes. Recent evidence suggests that increased p53 activity can, at least under some circumstances, promote organismal aging. Here, we discuss the role of p53 as a key regulator of the DNA damage responses, and discuss how p53 integrates the outcome of the DNA damage response to optimally balance tumor suppression and longevity.

Impact of genetic background and experimental reproducibility on identifying chemical compounds with robust longevity effects.

Limiting the debilitating consequences of ageing is a major medical challenge of our time. Robust pharmacological interventions that promote healthy ageing across diverse genetic backgrounds may engage conserved longevity pathways. Here we report results from the Caenorhabditis Intervention Testing Program in assessing longevity variation across 22 Caenorhabditis strains spanning 3 species, using multiple replicates collected across three independent laboratories. Reproducibility between test sites is high, whereas individual trial reproducibility is relatively low. Of ten pro-longevity chemicals tested, six significantly extend lifespan in at least one strain. Three reported dietary restriction mimetics are mainly effective across C. elegans strains, indicating species and strain-specific responses. In contrast, the amyloid dye ThioflavinT is both potent and robust across the strains. Our results highlight promising pharmacological leads and demonstrate the importance of assessing lifespans of discrete cohorts across repeat studies to capture biological variation in the search for reproducible ageing interventions.

A Caenorhabditis elegans Model Elucidates a Conserved Role for TRPA1-Nrf Signaling in Reactive α-Dicarbonyl Detoxification.

Reactive α-dicarbonyls (α-DCs), like methylglyoxal (MGO), accumulate with age and have been implicated in aging and various age-associated pathologies, such as diabetic complications and neurodegenerative disorders like Alzheimer's and Parkinson's diseases. Evolutionarily conserved glyoxalases are responsible for α-DC detoxification; however, their core biochemical regulation has remained unclear. We have established a Caenorhabditis elegans model, based on an impaired glyoxalase (glod-4/GLO1), to broadly study α-DC-related stress. We show that, in comparison to wild-type (N2, Bristol), glod-4 animals rapidly exhibit several pathogenic phenotypes, including hyperesthesia, neuronal damage, reduced motility, and early mortality. We further demonstrate TRPA-1/TRPA1 as a sensor for α-DCs, conserved between worms and mammals. Moreover, TRPA-1 activates SKN-1/Nrf via calcium-modulated kinase signaling, ultimately regulating the glutathione-dependent (GLO1) and co-factor-independent (DJ1) glyoxalases to detoxify α-DCs. Interestingly, this pathway is in stark contrast to the TRPA-1 activation and the ensuing calcium flux implicated in cold sensation in C. elegans, whereby DAF-16/FOXO gets activated via complementary kinase signaling. Finally, a phenotypic drug screen using C. elegans identified podocarpic acid as a novel activator of TRPA1 that rescues α-DC-induced pathologies in C. elegans and mammalian cells. Our work thus identifies TRPA1 as a bona fide drug target for the amelioration of α-DC stress, which represents a viable option to address aging-related pathologies in diabetes and neurodegenerative diseases.

Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Response Pathway Genes skn-1, ire-1, and xbp-1.

Vitamin D has multiple roles, including the regulation of bone and calcium homeostasis. Deficiency of 25-hydroxyvitamin D, the major circulating form of vitamin D, is associated with an increased risk of age-related chronic diseases, including Alzheimer's disease, Parkinson's disease, cognitive impairment, and cancer. In this study, we utilized Caenorhabditis elegans to examine the mechanism by which vitamin D influences aging. We found that vitamin-D3-induced lifespan extension requires the stress response pathway genes skn-1, ire-1, and xbp-1. Vitamin D3 (D3) induced expression of SKN-1 target genes but not canonical targets of XBP-1. D3 suppressed an important molecular pathology of aging, that of widespread protein insolubility, and prevented toxicity caused by human ß-amyloid. Our observation that D3 improves protein homeostasis and slows aging highlights the importance of maintaining appropriate vitamin D serum levels and may explain why such a wide variety of human age-related diseases are associated with vitamin D deficiency.

A DNA synthesis inhibitor is protective against proteotoxic stressors via modulation of fertility pathways in Caenorhabditis elegans.

Loss of germline precursor cells in C. elegans has previously been shown to improve protein homeostasis and extend lifespan, possibly due to reallocation of resources to somatic cells. In contrast, mutants that are sterile simply due to loss of sperm or oocyte production have a normal lifespan, often leading to the conclusion that loss of reproduction per se may have minor effects on C. elegans. We have found that inhibiting reproduction in C. elegans via the DNA synthesis inhibitor 5-fluoro-2-deoxyuridine (FUdR) improves protein homeostasis, stress resistance, and healthspan in wild-type animals. We find that FUdR is dependent on oogenesis and oocytic maturation. The effects of FUdR are dependent on FEM pathways, which regulate initiation of spermatogenesis. Loss of FEM expression leads to feminized animals that maintain arrested oocytes and are refractory to the effects of FUdR. FUdR-dependence is restored by spermatogenic signals, which trigger oocytic maturation and ovulation. Further, loss of FEM-3, a novel protein required for spermatogenesis, is sufficient to improve aspects of proteostasis. These effects are independent of previously described germline signals, including the DAF-16/FOXO, DAF-12/VDR, and HSF-1 pathways. These findings suggest that genetic or chemical inhibition of oocyte production can improve protein homeostasis in C. elegans.

Age-related micro-RNA abundance in individual C. elegans.

Non-coding small RNAs of the micro-RNA class (miRNA) are conserved regulators of gene function with a broad impact on biological processes. We screened miRNA levels for age-related changes in individual worms and investigated their influence on the lifespan of the nematode C. elegans. We measured the abundance of 69 miRNAs expressed in individual animals at different ages with over thirty five thousand discrete quantitative nano-fluidic polymerase chain reactions. We found that miRNA abundance was highly variable between individual worms raised under identical conditions and that expression variability generally increased with age. To identify expression differences associated with either reproductive or somatic tissues, we analyzed wild type and mutants that lacked germlines. miRNAs from the mir-35-41 cluster increased in abundance with age in wild type animals, but were nearly absent from mutants lacking a germline, suggesting their age-related increase originates from the germline. Most miRNAs with age-dependent levels did not have a major effect on lifespan, as corresponding deletion mutants exhibited wild-type lifespans. The major exception to this was mir-71, which increased in abundance with age and was required for normal longevity. Our genetic characterization indicates that mir-71 acts at least partly in parallel to insulin/IGF like signals to influence lifespan.

MicroRNAs miR-146a/b negatively modulate the senescence-associated inflammatory mediators IL-6 and IL-8.

Senescence is a cellular program that irreversibly arrests the proliferation of damaged cells and induces the secretion of the inflammatory mediators IL- 6 and IL-8 which are part of a larger senescence associated secretory phenotype (SASP). We screened quiescent and senescent human fibroblasts for differentially expressed microRNAS (miRNAs) and found that miRNAs 146a and 146b (miR-146a/b) were significantly elevated during senescence. We suggest that delayed miR-146a/b induction might be a compensatory response to restrain inflammation. Indeed, ectopic expression of miR-146a/b in primary human fibroblasts suppressed IL-6 and IL-8 secretion and downregulated IRAK1, a crucial component of the IL-1 receptor signal transduction pathway. Cells undergoing senescence without induction of a robust SASP did not express miR-146a/b. Further, IL-1alpha neutralizing antibodies abolished both miR-146a/b expression and IL-6 secretion. Our findings expand the biological contexts in which miRNA-146a/b modulates inflammatory responses. They suggest that IL-1 receptor signaling initiates both miR-146a/b upregulation and cytokine secretion, and that miR-146a/b is expressed in response to rising inflammatory cytokine levels as part of a negative feedback loop that restrains excessive SASP activity.

Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b.

Deregulation of micro-RNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a and miR-125b within the 3'-untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR-125a or miR-125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3' 3'-untranslated regions of ERBB2 and ERBB3 demonstrated approximately 35% less activity in miR-125a- and miR-125b-expressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing either miR-125a or miR-125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR-125a-or miR-125b-overexpressing SKBR3 cells were impaired in their anchorage-dependent growth and exhibited reduced migration and invasion capacities. Parallel studies performed on MCF10A cells demonstrated that miR-125a or miR-125b overexpression produced only marginal influences on the growth and migration of these non-transformed human mammary epithelial cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression and function.