Mitochondrial Dysfunction-associated Senescence Research Articles

Abstract 5861: CDK4/6 therapy-induced senescence as a resistance mechanism-MiDAS touch

Abstract Background: Cyclin-dependent kinase inhibitors (CDK4/6i) have revolutionized the therapy for estrogen receptor positive (ER+) breast cancer. Despite the response to CDK4/6i, resistance and recurrence are still a clinical reality, resulting in death due to breast cancer. G1 cell cycle arrest has been considered as the main mechanism of action of CDK4/6i in luminal breast cancer. However, the response to therapy does not correlate with the expression of CDK4/6 or key cell cycle proteins. It is also unclear whether CDK4/6i-induced senescence enhances the therapeutic response or mediates development of resistance. Experimental Procedures: We have developed drug-tolerant persisters resistant to CDK4/6 inhibitors (CDK4/6i-DTPs) in a panel of estrogen receptor positive (ER+) cells to explore the molecular mechanisms associated with resistance. Using different cell phenotype assays (i.e., cell viability/cell cycle, senescence/senescence reversibility and wound healing) as well as senescence-associated secretory phenotype (SASP) human cytokine array of 105 markers, we characterized the phenotype and secretome of these CDK4/6i DTPs. Summary: CDK4/6i-DTPs induced by abemaciclib, palbociclib and ribociclib (all from Selleckchem) demonstrated resistance to these drugs and dramatically slowed growth. The percentage of senescent cells reached 30-89% of the total population for all cell lines in all three drugs, whereas control parental cells exhibited around 10%. CDK4/6i DTPs also exhibited significant cell migration, and increased lysosomal phenotype compared to their parental sensitive cells. Further analysis with human cytokine array revealed that protein levels of growth differentiation factor 15 (GDF-15) are elevated in both cell lysates and conditioned-media of CDK4/6i DTPs, whereas Serpin E1 (Plasminogen Activator Inhibitor, Type I, PAI-1) was unique to secreted CDK4/6i DTPs. Interestingly, we did not observe changes in pro-inflammatory senescence-associated secretory phenotype (SASP) markers such as interleukins. Apart from being a SASP protein, GDF-15 is a mitochondrial cytokine (mitokine), suggesting a role in mitochondrial dysfunction-associated senescence (MiDAS). HMGB1, and LMNB1 are also down-regulated in CDK4/6-DTPs supporting the MiDAS phenotype. CDK4/6i-induced MiDAS appears to be transient, as CDK4/6i-DTPs reverted to the parental phenotype in drug-free culture media 20 days after the removal of CDK4/6i's. This included growth characteristics and sensitivity to CDK4/6i. This reversion of phenotype was observed in all five cell line models tested with all three drugs. Conclusions: CDK4/6-induces a mitochondrial dysfunction-associated senescence (MiDAS). This appears to be transient in nature as withdrawal of the drug results reacquisition of parental cell phenotype and drug-sensitivity. Targeting MiDAS pathway might be critical to improve efficacy of CDK4/6 inhibitors. Citation Format: Yesim Gokmen-Polar, Yuan Gu, Ling Ruan, Sunil S. Badve. CDK4/6 therapy-induced senescence as a resistance mechanism-MiDAS touch [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5861.

Changes in AMPK activity induces cellular senescence in human dental follicle cells

Dental Follicle Cells (DFCs) are somatic stem cells with a limited lifespan, but little is known about a possible mechanism of cellular senescence. Previous studies have shown that cellular senescence is associated with increased demand of glycolsis or the “glycolytic metabotype”, which can be induced by activation of 5′ adenosine monophosphate-activated protein kinase (AMPK), and decreased autophagy. This study examined the role of AMPK in inducing senescence in DFCs. During the induction of cellular senescence, AMPK activity was impaired, suggesting a negative impact on senescence induction. In line with this assumption, cellular senescence was induced upon inhibition of AMPK with a specific siRNA. In addition, after this inhibition, autophagy was also inhibited. Moreover, specific inhibition of autophagy promoted cellular senescence. However, inducers of AMPK such as metformin or AICAR surprisingly increased senescence in DFCs. Interestingly, autophagy was impaired after long-term induction of AMPK with AICAR and metformin. Moreover, activation of AMPK induces the consumption of glucose but decreases NAD/NADH ratio in DFCs that suggest not only “glycolytic metabotype” of DFCs but also Mitochondrial Dysfunction Associated Senescence (MiDAS). Both changes are highly associated with the induction of cellular senescence. Hence, both AMPK activation and inhibition promote the induction of cellular senecence of DFCs.

AtFAHD1a: A New Player Influencing Seed Longevity and Dormancy in Arabidopsis?

Fumarylacetoacetate hydrolase (FAH) proteins form a superfamily found in Archaea, Bacteria, and Eukaryota. However, few fumarylacetoacetate hydrolase domain (FAHD)-containing proteins have been studied in Metazoa and their role in plants remains elusive. Sequence alignments revealed high homology between two Arabidopsis thaliana FAHD-containing proteins and human FAHD1 (hFAHD1) implicated in mitochondrial dysfunction-associated senescence. Transcripts of the closest hFAHD1 orthologue in Arabidopsis (AtFAHD1a) peak during seed maturation drying, which influences seed longevity and dormancy. Here, a homology study was conducted to assess if AtFAHD1a contributes to seed longevity and vigour. We found that an A. thaliana T-DNA insertional line (Atfahd1a-1) had extended seed longevity and shallower thermo-dormancy. Compared to the wild type, metabolite profiling of dry Atfahd1a-1 seeds showed that the concentrations of several amino acids, some reducing monosaccharides, and δ-tocopherol dropped, whereas the concentrations of dehydroascorbate, its catabolic intermediate threonic acid, and ascorbate accumulated. Furthermore, the redox state of the glutathione disulphide/glutathione couple shifted towards a more reducing state in dry mature Atfahd1a-1 seeds, suggesting that AtFAHD1a affects antioxidant redox poise during seed development. In summary, AtFAHD1a appears to be involved in seed redox regulation and to affect seed quality traits such as seed thermo-dormancy and longevity.

Twist1 regulates cellular senescence in mesenchymal stem cells

Purpose: Mesenchymal stem cells (MSCs) are promising cells to treat skeletal related diseases such as osteoarthritis (OA). One of the limitations that reduces the therapeutic use of MSCs for OA cell-based treatments is cellular senescence. Cellular senescence is a state of the cell in which cells stop proliferating, while still be metabolically active. By acquiring a specific senescence-associated secretory phenotype (SASP), senescent MSCs can contribute to cartilage degeneration and chronic inflammation. The molecular mechanism behind cellular senescence in MSCs remains largely unknown. The transcription factor TWIST1 is suggested to play a role in cellular senescence, since its expression is reduced in slow proliferating MSCs and we previously demonstrated that high TWIST1-expressing MSCs have an enhanced expansion capacity. The aim of this research is therefore to determine the role of TWIST1 in regulating cellular senescence of MSCs. Methods: Human MSCs were obtained from bone chips from leftover iliac crest of patients undergoing cleft palate reconstructive surgery (N=4, 9-13 years). MSCs were expanded in alpha-MEM containing 10% serum and 1 ng/ml FGF2. To determine the effect of TWIST1 silencing on senescence, MSCs were treated with 15 nM TWIST1 siRNA and Lipofectamine RNAMAX Transfection Reagent (siTWIST1). Scramble siRNA and untreated cells were used as controls. To overexpress TWIST1, MSCs were transduced with a third generation TWIST1 overexpression lentiviral construct. Untreated MSCs and MSCs which overexpress GFP or an empty vector were used as control. The transduction efficiency, of GFP-positive MSCs, was determined using fluorescent microscopy. To determine cellular senescence, we determined senescence-associated beta-galactosidase (SA-β-gal) activity and quantified negative, low and high positive cells. In addition gene expression for IL8, IL6, IL1B, P21 and P16 were analyzed to assess cellular senescence in MSCs. Gene expression was normalized using the best housekeeper index of HPRT, RSP27a and GAPDH. The expansion capacity of the MSCs was determined by cell count. To analyze statistical significance, linear mixed model was applied, the different conditions were considered as fixed parameter and the donors as random parameter. Bonferroni post hoc test was performed to correct for multiple comparisons. Results: TWIST1 transduced MSCs had an average of 151-fold increase in TWIST1 expression detected by RT-PCR, a reduced percentage of SA-B-gal positive cells (71% reduction) and a reduced expression of the SASP related genes IL6 (1.8-fold reduction), IL8 (2.4-fold reduction) and IL1B (1.3-fold reduction) compared to transduction control (Figure 1). In addition, the expansion capacity of MSCs overexpressing TWIST1 was increased, resulting in a 1.5-fold increase in cell number after 4 days. These data indicate that TWIST1 delays cellular senescence in MSCs. Next, we determined the effect of TWIST1 silencing on cellular senescence associated markers. A high percentage of siTWIST1 MSCs were high positive for SA-B-gal (9.5-fold compared to controls, Figure 2A) and cells had a reduced expansion capacity compared to control conditions (20-fold reduction in cell number after 14 days). These data suggest that silencing TWIST1 in MSCs increases cellular senescence. Surprisingly, the expression of SASP related genes IL6 and IL8 was reduced in siTWIST cells compared to untreated control (IL6, 1.6-fold; IL8, 7.2-fold reduction, Figure 2B) and no induction of IL1B was observed in the siTWIST1 MSCs. Low IL6, IL8 and IL1B expression in senescent cells was recently described as mitochondrial dysfunction-associated senescence (MiDAS). We then further analyzed siTWIST1 MSCs for the expression of senescent markers not associated with a SASP phenotype and found that P16 and P21, associated to the cell cycle regulation, were increased after TWIST1 silencing (5.9-fold and 1.6-fold, respectively) in accordance with a senescent phenotype. Overall, these data indicate that TWIST1 silencing results in cellular senescence of MSCs with an uncommon SASP expression. Conclusions: Our Results indicate that TWIST1 plays a role in a special type of cellular senescence, previously described as MiDAS, with a unique SASP expression profile, lacking IL6, IL8 and IL1B upregulation. In addition these data show that different type of senescent cells can have a different SASP expression and therefore potentially have different effects on the microenvironment. Here, we show how TWIST1 may be a target to modulate cellular senescence to improve MSC-based OA therapies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Oxaloacetate decarboxylase FAHD1 – a new regulator of mitochondrial function and senescence

FAHD1, a member of the FAH superfamily of enzymes, was identified in a proteomic screen for mitochondrial proteins with differential expression in young versus senescent human endothelial cells. FAHD1 acts as oxaloacetate decarboxylase, and recent observations suggest that FAHD1 plays an important role in regulating mitochondrial function. Thus, mutation of the nematode homolog, fahd-1, impairs mitochondrial function in Caenorhabditis elegans. When FAHD1 gene expression was silenced in human cells, activity of the mitochondrial electron transport (ETC) system was reduced and the cells entered premature senescence-like growth arrest. These findings suggest a model where FAHD1 regulates mitochondrial function and in consequence senescence. These findings are discussed here in the context of a new concept where senescence is divided into deep senescence and less severe forms of senescence. We propose that genetic inactivation of FAHD1 in human cells induces a specific form of cellular senescence, which we term senescence light and discuss it in the context of mitochondrial dysfunction associated senescence (MiDAS) described by others. Together these findings suggest the existence of a continuum of cellular senescence phenotypes, which may be at least in part reversible.

Maternal high calorie diet induces mitochondrial dysfunction and senescence phenotype in subcutaneous fat of newborn mice.

Mitochondrial dysfunction, inflammation and senescence-like features are observed in adipose depots in aging and obesity. Herein, we evaluated how maternal high calorie diet (HCD) may impact on subcutaneous adipose tissue (sAT) of the newborn mice. Adult C57BL/6J mice were randomly divided in three groups: normal calorie diet (NCD), HCD and HCD supplemented with niacin 8 weeks before mating. Mothers and pups were then sacrificed and metabolic and molecular analyses were carried out on sAT. HCD induced mitochondria dysfunction in mothers without inflammation and senescence, whereas in pups we also revealed the occurrence of senescent phenotype. The mitochondrial dysfunction-associated senescence in pups was accompanied by a drop in NAD+/NADH ratio and alteration in the NAD+-dependent enzymes PARP1 and SIRT1. Importantly, maternal dietary supplementation with niacin during gestation and lactation restrained NAD+/NADH decrease imposed by HCD limiting inflammatory cytokine production and senescence phenotype in newborn sAT. Given the fundamental role of sAT in buffering nutrient overload and avoiding pathogenic ectopic fat accumulation, we suggest that NAD+ boosting strategies during maternal HCD could be helpful in limiting sAT dysfunction in newborn.

Mitochondrial Dysfunction Meets Senescence

Cellular senescence and mitochondrial dysfunction are hallmarks of ageing, but until now their relationship has not been clear. Recent work by Wiley et al. shows that mitochondrial defects can cause a distinct senescence phenotype termed MiDAS (mitochondrial dysfunction-associated senescence). MiDAS has a specific secretome that is able to drive some of the aging phenotypes. These findings suggest novel therapeutic opportunities for treating age-related pathologies.

Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype

Cellular senescence permanently arrests cell proliferation, often accompanied by a multi-faceted senescence-associated secretory phenotype (SASP). Loss of mitochondrial function can drive age-related declines in the function of many post-mitotic tissues, but little is known about how mitochondrial dysfunction affects mitotic tissues. We show here that several manipulations that compromise mitochondrial function in proliferating human cells induce a senescence growth arrest with a modified SASP that lacks the IL-1-dependent inflammatory arm. Cells that underwent mitochondrial dysfunction-associated senescence (MiDAS) had lower NAD+/NADH ratios, which caused both the growth arrest and prevented the IL-1-associated SASP through AMPK-mediated p53 activation. Progeroid mice that rapidly accrue mtDNA mutations accumulated senescent cells with a MiDAS SASP in vivo, which suppressed adipogenesis and stimulated keratinocyte differentiation in cell culture. Our data identify a distinct senescence response and provide a mechanism by which mitochondrial dysfunction can drive aging phenotypes.