Genetic interrogation of replicative senescence uncovers a dual role for USP28 in coordinating the p53 and GATA4 branches of the senescence program.

Senescence is a terminal differentiation program that halts the growth of damaged cells and must be circumvented for cancer to arise. Here we describe a panel of genetic screens to identify genes required for replicative senescence. We uncover a role in senescence for the potent tumor suppressor and ATM substrate USP28. USP28 controls activation of both the TP53 branch and the GATA4/NFkB branch that controls the senescence-associated secretory phenotype (SASP). These results suggest a role for ubiquitination in senescence and imply a common node downstream from ATM that links the TP53 and GATA4 branches of the senescence response.

A gain-of-function senescence bypass screen identifies the homeobox transcription factor DLX2 as a regulator of ATM-p53 signaling.

Senescence stimuli activate multiple tumor suppressor pathways to initiate cycle arrest and a differentiation program characteristic of senescent cells. We performed a two-stage, gain-of-function screen to select for the genes whose enhanced expression can bypass replicative senescence. We uncovered multiple genes known to be involved in p53 and Rb regulation and ATM regulation, two components of the CST (CTC1-STN1-TEN1) complex involved in preventing telomere erosion, and genes such as REST and FOXO4 that have been implicated in aging. Among the new genes now implicated in senescence, we identified DLX2, a homeobox transcription factor that has been shown to be required for tumor growth and metastasis and is associated with poor cancer prognosis. Growth analysis showed that DLX2 expression led to increased cellular replicative life span. Our data suggest that DLX2 expression reduces the protein components of the TTI1/TTI2/TEL2 complex, a key complex required for the proper folding and stabilization of ATM and other members of the PIKK (phosphatidylinositol 3-kinase-related kinase) family kinase, leading to reduced ATM-p53 signaling and senescence bypass. We also found that the overexpression of DLX2 exhibited a mutually exclusive relationship with p53 alterations in cancer patients. Our functional screen identified novel players that may promote tumorigenesis by regulating the ATM-p53 pathway and senescence.

Combined Fractional Treatment of Acne Scars Involving Non-ablative 1,550-nm Erbium-glass Laser and Micro-needling Radiofrequency: A 16-week Prospective, Randomized Split-face Study.

An optimized therapeutic regimen involving a non-ablative fractionated laser or radiofrequency therapy for acne scars has not yet been established. To evaluate whether the combination of a non-ablative fractional laser (NAF) and fractional micro-needling radiofrequency (FMR) has clinical advantages for the treatment of atrophic acne scars compared with NAF alone, a 16-week prospective, randomized split-face study was performed. Each facial side of a patient was treated with 3 sessions of either NAF with FMR or NAF alone, with a 4-week interval between each session. Although both sides demonstrated significant decreases in the échelle d'évaluation clinique des cicatrices d'acné (ECCA) score, the facial side treated using the combination regimen demonstrated greater improvement in ECCA score regarding degree and onset time than the NAF-treated side. Histopathological and immunohistochemical results confirmed the clinical findings. This study demonstrated that a combination regimen involving NAF and FMR could be a viable option with satisfactory efficacy.

Systemic regulation of starvation response in Caenorhabditis elegans.

When the supply of environmental nutrients is limited, multicellular animals can make both physiological and behavioral changes so as to cope with nutrient starvation. Although physiological and behavioral effects of starvation are well known, the mechanisms by which animals sense starvation systemically remain elusive. Furthermore, what constituent of food is sensed and how it modulates starvation response is still poorly understood. In this study, we use a starvation-hypersensitive mutant to identify molecules and mechanisms that modulate starvation signaling. We found that specific amino acids could suppress the starvation-induced death of gpb-2 mutants, and that MGL-1 and MGL-2, Caenorhabditis elegans homologs of metabotropic glutamate receptors, were involved. MGL-1 and MGL-2 acted in AIY and AIB neurons, respectively. Treatment with leucine suppressed starvation-induced stress resistance and life span extension in wild-type worms, and mutation of mgl-1 and mgl-2 abolished these effects of leucine. Taken together, our results suggest that metabotropic glutamate receptor homologs in AIY and AIB neuron may modulate a systemic starvation response, and that C. elegans senses specific amino acids as an anti-hunger signal.

Living Beyond Restriction: LBR promotes cellular immortalization by suppressing genomic instability and senescence.

Cellular immortalization is a complex process that requires multiple genetic alterations to overcome restricting barriers, including senescence. Not surprisingly, many of these alterations are associated with cancer; two tumor suppressor pathways, the cellular tumor antigen p53 and p16-Retinoblastoma (RB) pathways, are the best-characterized examples, but their mutations alone are known to be insufficient to drive full immortalization. En et al. identified a role for the lamin B receptor (LBR) in promoting cellular proliferation and immortalization in p53- and RB-deficient cells by maintaining their genome integrity and suppressing senescence. Thus, modulation of LBR could be exploited to treat cancer and potentially also to promote cell rejuvenation.

Metabolic remodeling in cancer and senescence and its therapeutic implications.

Cellular metabolism is a flexible and plastic network that often dictates physiological and pathological states of the cell, including differentiation, cancer, and aging. Recent advances in cancer metabolism represent a tremendous opportunity to treat cancer by targeting its altered metabolism. Interestingly, despite their stable growth arrest, senescent cells - a critical component of the aging process - undergo metabolic changes similar to cancer metabolism. A deeper understanding of the similarities and differences between these disparate pathological conditions will help identify which metabolic reprogramming is most relevant to the therapeutic liabilities of senescence. Here, we compare and contrast cancer and senescence metabolism and discuss how metabolic therapies can be established as a new modality of senotherapy for healthy aging.

Brief guide to senescence assays using cultured mammalian cells.

Cellular senescence is a crucial biological process associated with organismal aging and many chronic diseases. Here, we present a brief guide to mammalian senescence assays, including the measurement of cell cycle arrest, change in cellular morphology, senescence-associated ß-galactosidase (SA-ß-gal) staining, and the expression of senescence-associated secretory phenotype (SASP). This work will be useful for biologists with minimum expertise in cellular senescence assays.

Where should siRNAs go: applicable organs for siRNA drugs.

RNA interference mediated by small interfering RNAs (siRNAs) has been exploited for the development of therapeutics. siRNAs can be a powerful therapeutic tool because the working mechanisms of siRNAs are straightforward. siRNAs determine targets based on their sequence and specifically regulate the gene expression of the target gene. However, efficient delivery of siRNAs to the target organ has long been an issue that needs to be solved. Tremendous efforts regarding siRNA delivery have led to significant progress in siRNA drug development, and from 2018 to 2022, a total of five siRNA drugs were approved for the treatment of patients. Although all FDA-approved siRNA drugs target the hepatocytes of the liver, siRNA-based drugs targeting different organs are in clinical trials. In this review, we introduce siRNA drugs in the market and siRNA drug candidates in clinical trials that target cells in multiple organs. The liver, eye, and skin are the preferred organs targeted by siRNAs. Three or more siRNA drug candidates are in phase 2 or 3 clinical trials to suppress gene expression in these preferred organs. On the other hand, the lungs, kidneys, and brain are challenging organs with relatively few clinical trials. We discuss the characteristics of each organ related to the advantages and disadvantages of siRNA drug targeting and strategies to overcome the barriers in delivering siRNAs based on organ-specific siRNA drugs that have progressed to clinical trials.

Lysosomal control of senescence and inflammation through cholesterol partitioning.

Whereas cholesterol is vital for cell growth, proliferation, and remodeling, dysregulation of cholesterol metabolism is associated with multiple age-related pathologies. Here we show that senescent cells accumulate cholesterol in lysosomes to maintain the senescence-associated secretory phenotype (SASP). We find that induction of cellular senescence by diverse triggers enhances cellular cholesterol metabolism. Senescence is associated with the upregulation of the cholesterol exporter ABCA1, which is rerouted to the lysosome, where it moonlights as a cholesterol importer. Lysosomal cholesterol accumulation results in the formation of cholesterol-rich microdomains on the lysosomal limiting membrane enriched with the mammalian target of rapamycin complex 1 (mTORC1) scaffolding complex, thereby sustaining mTORC1 activity to support the SASP. We further show that pharmacological modulation of lysosomal cholesterol partitioning alters senescence-associated inflammation and in vivo senescence during osteoarthritis progression in male mice. Our study reveals a potential unifying theme for the role of cholesterol in the aging process through the regulation of senescence-associated inflammation.

A focused natural compound screen reveals senolytic and senostatic effects of Isatis tinctoria.

Natural products and their derivatives historically represent alternatives to conventional synthetic molecules for pharmacotherapy, ranging from cancer chemotherapeutics to cosmetic ingredients that exert anti-aging activities. Cellular senescence is considered a main driver of skin aging, yet natural products that target skin senescence in a specific manner are not thoroughly explored. Here, we performed a focused compound screen to identify natural products that exert anti-senescence effects. We found that Isatis tinctoria, woad extracts, displayed a senolytic effect on senescent human skin fibroblasts. Furthermore, treatment with woad extracts attenuated the expression of pro-inflammatory senescence-associated secretory phenotype (SASP), showing a senostatic activity. Intriguingly, woad extracts displayed only a marginal cytotoxic effect toward senescent human lung fibroblasts. Thus, our results reveal the potential activities of woad extracts for targeting skin senescence and suggest that woad extracts could be an attractive ingredient for cosmetics to prevent skin aging.

Functional role of the Frizzled linker domain in the Wnt signaling pathway.

The Wnt signaling pathway plays a critical role in the developmental and physiological processes of metazoans. We previously reported that the Frizzled4 (FZD4) linker domain plays an important role in Norrin binding and signaling. However, the question remains whether the FZD linker contributes to Wnt signaling in general. Here, we show that the FZD linker is involved in Wnt binding and affects downstream Wnt signaling. A FZD4 chimera, in which the linker was swapped with that of the non-canonical receptor FZD6, impairs the binding with WNT3A and suppresses the recruitment of LRP6 and Disheveled, resulting in reduced canonical signaling. A similar effect was observed for non-canonical signaling. A FZD6 chimera containing the FZD1 linker showed reduced WNT5A binding and impaired signaling in ERK, JNK, and AKT mediated pathways. Altogether, our results suggest that the FZD linker plays an important role in specific Wnt binding and intracellular Wnt signaling.

The FMRFamide Neuropeptide FLP-20 Acts as a Systemic Signal for Starvation Responses in Caenorhabditis elegans.

Most animals face frequent periods of starvation throughout their entire life and thus need to appropriately adjust their behavior and metabolism during starvation for their survival. Such adaptive responses are regulated by a complex set of systemic signals, including hormones and neuropeptides. While much progress has been made in identifying pathways that regulate nutrient-excessive states, it is still incompletely understood how animals systemically signal their nutrient-deficient states. Here, we showed that the FMRFamide neuropeptide FLP-20 modulates a systemic starvation response in Caenorhabditis elegans. We found that mutation of flp-20 rescued the starvation hypersensitivity of the G protein ß-subunit gpb-2 mutants by suppressing excessive autophagy. FLP-20 acted in AIB neurons, where the metabotropic glutamate receptor MGL-2 also functions to modulate a systemic starvation response. Furthermore, FLP-20 modulated starvation-induced fat degradation in a manner dependent on the receptor-type guanylate cyclase GCY-28. Collectively, our results reveal a circuit that senses and signals nutrient-deficient states to modulate a systemic starvation response in multicellular organisms.

Targeting the stress support network regulated by autophagy and senescence for cancer treatment.

Autophagy and cellular senescence are two potent tumor suppressive mechanisms activated by various cellular stresses, including the expression of activated oncogenes. However, emerging evidence has also indicated their pro-tumorigenic activities, strengthening the case for the complexity of tumorigenesis. More specifically, tumorigenesis is a systemic process emanating from the combined accumulation of changes in the tumor support pathways, many of which cannot cause cancer on their own but might still provide excellent therapeutic targets for cancer treatment. In this review, we discuss the dual roles of autophagy and senescence during tumorigenesis, with a specific focus on the stress support networks in cancer cells modulated by these processes. A deeper understanding of such context-dependent roles may help to enhance the effectiveness of cancer therapies targeting autophagy and senescence, while limiting their potential side effects. This will steer and accelerate the pace of research and drug development for cancer treatment.

All cells are created equal in the sight of autophagy: selective autophagy maintains homeostasis in senescent cells.

Macroautophagy/autophagy is a sophisticated quality control program that limits cellular damage and maintains homeostasis, being an essential part of several lifespan-promoting interventions. However, autophagy is also necessary for full establishment of cellular senescence, a causal factor for many age-related diseases and aging. What lies ahead of us to unravel such a paradoxical role of autophagy in senescence is to identify specific targets degraded by autophagy during senescence and determine their importance in the senescence regulatory network. Recently, we developed the "Selective autophagy substrates Identification Platform (SIP)" to advance these goals, providing a rich set of autophagy substrate proteins involved in senescence. Our study demonstrated that selective autophagy coordinates the stress support networks in senescent cells by degrading multiple regulatory components, echoing its homeostatic roles in normal cells. Targeting this type of selective autophagy might provide a unique opportunity to develop non-senescence addiction-based therapeutic strategies for senotherapy by disturbing the homeostatic state of senescent cells.

A flow-cytometry-based assessment of global protein synthesis in human senescent cells.

Senescent cells constantly experience stressful conditions and restrain their protein translation to cope with it. Here, we present a detailed protocol to measure the rate of global protein synthesis using L-azidohomoalanine (L-AHA)-based click chemistry in human senescent fibroblasts. We optimized several aspects of the procedure, including senescence induction, a flow cytometry analysis of senescent cells, and the duration of L-AHA incorporation. This protocol uses senescent human fibroblasts but can be applied to other types of cells or circumstances. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).

UXT chaperone prevents proteotoxicity by acting as an autophagy adaptor for p62-dependent aggrephagy.

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.

Coordinate regulation of the senescent state by selective autophagy.

Cellular senescence is a complex stress response implicated in aging. Autophagy can suppress senescence but is counterintuitively necessary for full senescence. Although its anti-senescence role is well described, to what extent autophagy contributes to senescence establishment and the underlying mechanisms is poorly understood. Here, we show that selective autophagy of multiple regulatory components coordinates the homeostatic state of senescence. We combined a proteomic analysis of autophagy components with protein stability profiling, identifying autophagy substrate proteins involved in several senescence-related processes. Selective autophagy of KEAP1 promoted redox homeostasis during senescence. Furthermore, selective autophagy limited translational machinery components to ameliorate senescence-associated proteotoxic stress. Lastly, selective autophagy of TNIP1 enhanced senescence-associated inflammation. These selective autophagy networks appear to operate in vivo senescence during human osteoarthritis. Our data highlight a caretaker role of autophagy in the stress support network of senescence through regulated protein stability and unravel the intertwined relationship between two important age-related processes.

MON-2, a Golgi protein, mediates autophagy-dependent longevity in Caenorhabditis elegans.

The Golgi apparatus plays a central role in trafficking cargoes such as proteins and lipids. Defects in the Golgi apparatus lead to various diseases, but its role in organismal longevity is largely unknown. Using a quantitative proteomic approach, we found that a Golgi protein, MON-2, was up-regulated in long-lived Caenorhabditis elegans mutants with mitochondrial respiration defects and was required for their longevity. Similarly, we showed that DOP1/PAD-1, which acts with MON-2 to traffic macromolecules between the Golgi and endosome, contributed to the longevity of respiration mutants. Furthermore, we demonstrated that MON-2 was required for up-regulation of autophagy, a longevity-associated recycling process, by activating the Atg8 ortholog GABARAP/LGG-1 in C. elegans. Consistently, we showed that mammalian MON2 activated GABARAPL2 through physical interaction, which increased autophagic flux in mammalian cells. Thus, the evolutionarily conserved role of MON2 in trafficking between the Golgi and endosome is an integral part of autophagy-mediated longevity.

Sclerostin inhibits Wnt signaling through tandem interaction with two LRP6 ectodomains.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the ß-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2-SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.