p16High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy.

The ability of an organism to overcome infectious diseases has traditionally been linked to killing invading pathogens. Accumulating evidence, however, indicates that, apart from restricting pathogen loads, organismal survival is coupled to an additional yet poorly understood mechanism called disease tolerance. Here we report that p16High immune cells play a key role in establishing disease tolerance. We found that the FDA-approved BNT162b2 mRNA COVID-19 vaccine is a potent and rapid inducer of p16High immune subsets both in mice and humans. In turn, p16High immune cells were indispensable for counteracting different lethal conditions, including LPS-induced sepsis, acute SARS-CoV-2 infection and ionizing irradiation. Mechanistically, we propose that activation of TLR7 or a low physiological activity of STING is sufficient to induce p16High immune subset that, in turn, establishes a low adenosine environment and disease tolerance. Furthermore, containing these signals within a beneficial range by deleting MDA5 that appeared sufficient to maintain a low activity of STING, induces p16High immune cells and delays organ deterioration upon aging with improved healthspan. Our data highlight the beneficial role of p16High immune subsets in establishing a low adenosine environment and disease tolerance.

p16High senescence restricts cellular plasticity during somatic cell reprogramming.

Despite advances in four-factor (4F)-induced reprogramming (4FR) in vitro and in vivo, how 4FR interconnects with senescence remains largely under investigated. Here, using genetic and chemical approaches to manipulate senescent cells, we show that removal of p16High cells resulted in the 4FR of somatic cells into totipotent-like stem cells. These cells expressed markers of both pluripotency and the two-cell embryonic state, readily formed implantation-competent blastoids and, following morula aggregation, contributed to embryonic and extraembryonic lineages. We identified senescence-dependent regulation of nicotinamide N-methyltransferase as a key mechanism controlling the S-adenosyl-L-methionine levels during 4FR that was required for expression of the two-cell genes and acquisition of an extraembryonic potential. Importantly, a partial 4F epigenetic reprogramming in old mice was able to reverse several markers of liver aging only in conjunction with the depletion of p16High cells. Our results show that the presence of p16High senescent cells limits cell plasticity, whereas their depletion can promote a totipotent-like state and histopathological tissue rejuvenation during 4F reprogramming.

Eliminating Senescent Cells Can Promote Pulmonary Hypertension Development and Progression.

BACKGROUND: Senescent cells (SCs) are involved in proliferative disorders, but their role in pulmonary hypertension remains undefined. We investigated SCs in patients with pulmonary arterial hypertension and the role of SCs in animal pulmonary hypertension models. METHODS: We investigated senescence (p16, p21) and DNA damage (γ-H2AX, 53BP1) markers in patients with pulmonary arterial hypertension and murine models. We monitored p16 activation by luminescence imaging in p16-luciferase (p16LUC/+) knock-in mice. SC clearance was obtained by a suicide gene (p16 promoter-driven killer gene construct in p16-ATTAC mice), senolytic drugs (ABT263 and cell-permeable FOXO4-p53 interfering peptide [FOXO4-DRI]), and p16 inactivation in p16LUC/LUC mice. We investigated pulmonary hypertension in mice exposed to normoxia, chronic hypoxia, or hypoxia+Sugen, mice overexpressing the serotonin transporter (SM22-5-HTT+), and rats given monocrotaline. RESULTS: Patients with pulmonary arterial hypertension compared with controls exhibited high lung p16, p21, and γ-H2AX protein levels, with abundant vascular cells costained for p16, γ-H2AX, and 53BP1. Hypoxia increased thoracic bioluminescence in p16LUC/+ mice. In wild-type mice, hypoxia increased lung levels of senescence and DNA-damage markers, senescence-associated secretory phenotype components, and p16 staining of pulmonary endothelial cells (P-ECs, 30% of lung SCs in normoxia), and pulmonary artery smooth muscle cells. SC elimination by suicide gene or ABT263 increased the right ventricular systolic pressure and hypertrophy index, increased vessel remodeling (higher dividing proliferating cell nuclear antigen-stained vascular cell counts during both normoxia and hypoxia), and markedly decreased lung P-ECs. Pulmonary hemodynamic alterations and lung P-EC loss occurred in older p16LUC/LUC mice, wild-type mice exposed to Sugen or hypoxia+Sugen, and SM22-5-HTT+ mice given either ABT263 or FOXO4-DRI, compared with relevant controls. The severity of monocrotaline-induced pulmonary hypertension in rats was decreased slightly by ABT263 for 1 week but was aggravated at 3 weeks, with loss of P-ECs. CONCLUSIONS: Elimination of senescent P-ECs by senolytic interventions may worsen pulmonary hemodynamics. These results invite consideration of the potential impact on pulmonary vessels of strategies aimed at controlling cell senescence in various contexts.

Defined p16High Senescent Cell Types Are Indispensable for Mouse Healthspan.

The accumulation of senescent cells can drive many age-associated phenotypes and pathologies. Consequently, it has been proposed that removing senescent cells might extend lifespan. Here, we generated two knockin mouse models targeting the best-characterized marker of senescence, p16Ink4a. Using a genetic lineage tracing approach, we found that age-induced p16High senescence is a slow process that manifests around 10-12 months of age. The majority of p16High cells were vascular endothelial cells mostly in liver sinusoids (LSECs), and to lesser extent macrophages and adipocytes. In turn, continuous or acute elimination of p16High senescent cells disrupted blood-tissue barriers with subsequent liver and perivascular tissue fibrosis and health deterioration. Our data show that senescent LSECs are not replaced after removal and have important structural and functional roles in the aging organism. In turn, delaying senescence or replacement of senescent LSECs could represent a powerful tool in slowing down aging.

LSEC model of aging.

Data obtained from genetically modified mouse models suggest a detrimental role for p16High senescent cells in physiological aging and age-related pathologies. Our recent analysis of aging mice revealed a continuous and noticeable accumulation of liver sinusoid endothelial cells (LSECs) expressing numerous senescence markers, including p16. At early stage, senescent LSECs show an enhanced ability to clear macromolecular waste and toxins including oxidized LDL (oxLDL). Later in life, however, the efficiency of this important detoxifying function rapidly declines potentially due to increased endothelial thickness and senescence-induced silencing of scavenger receptors and endocytosis genes. This inability to detoxify toxins and macromolecular waste, which can be further exacerbated by increased intestinal leakiness with age, might be an important contributing factor to animal death. Here, we propose how LSEC senescence could serve as an endogenous clock that ultimately controls longevity and outline some of the possible approaches to extend the lifespan.

DNA Damage Signaling-Induced Cancer Cell Reprogramming as a Driver of Tumor Relapse.

Accumulating evidence supports the role of the DNA damage response (DDR) in the negative regulation of tumorigenesis. Here, we found that DDR signaling poises a series of epigenetic events, resulting in activation of pro-tumorigenic genes but can go as far as reactivation of the pluripotency gene OCT4. Loss of DNA methylation appears to be a key initiating event in DDR-dependent OCT4 locus reactivation although full reactivation required the presence of a driving oncogene, such as Myc and macroH2A downregulation. Using genetic-lineage-tracing experiments and an in situ labeling approach, we show that DDR-induced epigenetic reactivation of OCT4 regulates the resistance to chemotherapy and contributes to tumor relapse both in mouse and primary human cancers. In turn, deletion of OCT4 reverses chemoresistance and delays the relapse. Here, we uncovered an unexpected tumor-promoting role of DDR in cancer cell reprogramming, providing novel therapeutic entry points for cancer intervention strategies.

Tumor-Stroma Mechanics Coordinate Amino Acid Availability to Sustain Tumor Growth and Malignancy.

Dysregulation of extracellular matrix (ECM) deposition and cellular metabolism promotes tumor aggressiveness by sustaining the activity of key growth, invasion, and survival pathways. Yet mechanisms by which biophysical properties of ECM relate to metabolic processes and tumor progression remain undefined. In both cancer cells and carcinoma-associated fibroblasts (CAFs), we found that ECM stiffening mechanoactivates glycolysis and glutamine metabolism and thus coordinates non-essential amino acid flux within the tumor niche. Specifically, we demonstrate a metabolic crosstalk between CAF and cancer cells in which CAF-derived aspartate sustains cancer cell proliferation, while cancer cell-derived glutamate balances the redox state of CAFs to promote ECM remodeling. Collectively, our findings link mechanical stimuli to dysregulated tumor metabolism and thereby highlight a new metabolic network within tumors in which diverse fuel sources are used to promote growth and aggressiveness. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in cancer.

Matrix Stiffening and EGFR Cooperate to Promote the Collective Invasion of Cancer Cells.

In squamous cell carcinoma (SCC), tissue invasion by collectively invading cells requires physical forces applied by tumor cells on their surrounding extracellular matrix (ECM). Cancer-related ECM is composed of thick collagen bundles organized by carcinoma-associated fibroblasts (CAF) within the tumor stroma. Here, we show that SCC cell collective invasion is driven by the matrix-dependent mechano-sensitization of EGF signaling in cancer cells. Calcium (Ca2+) was a potent intracellular second messenger that drove actomyosin contractility. Tumor-derived matrix stiffness and EGFR signaling triggered increased intracellular Ca2+ through CaV1.1 expression in SCC cells. Blocking L-type calcium channel expression or activity using Ca2+ channel blockers verapamil and diltiazem reduced SCC cell collective invasion both in vitro and in vivo These results identify verapamil and diltiazem, two drugs long used in medical care, as novel therapeutic strategies to block the tumor-promoting activity of the tumor niche.Significance: This work demonstrates that calcium channels blockers verapamil and diltiazem inhibit mechano-sensitization of EGF-dependent cancer cell collective invasion, introducing potential clinical strategies against stromal-dependent collective invasion.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5229/F1.large.jpg Cancer Res; 78(18); 5229-42. ©2018 AACR.

Aged dominant negative p38α MAPK mice are resistant to age-dependent decline in adult-neurogenesis and context discrimination fear conditioning.

A major aspect of mammalian aging is the decline in functional competence of many self-renewing cell types, including adult-born neuronal precursors. Since age-related senescence of self-renewal occurs simultaneously with chronic up-regulation of the p38MAPKalpha (p38α) signaling pathway, we used the dominant negative mouse model for attenuated p38α activity (DN-p38αAF/+) in which Thr180 and Tyr182 are mutated (T→A/Y→F) to prevent phosphorylation activation (DN-p38αAF/+) and kinase activity. As a result, aged DN-p38αAF/+ mice are resistant to age-dependent decline in proliferation and regeneration of several peripheral tissue progenitors when compared to wild-type littermates. Aging is the major risk factor for non-inherited forms of Alzheimer's disease (AD); environmental and genetic risk factors that accelerate the senescence phenotype are thought to contribute to an individual's relative risk. In the present study, we evaluated aged DN-p38αAF/+ and wildtype littermates in a series of behavioral paradigms to test if p38α mutant mice exhibit altered baseline abnormalities in neurological reflexes, locomotion, anxiety-like behavior, and age-dependent cognitive decline. While aged DN-p38αAF/+ and wildtype littermates appear equal in all tested baseline neurological and behavioral parameters, DN-p38αAF/+ exhibit superior context discrimination fear conditioning. Context discrimination is a cognitive task that is supported by proliferation and differentiation of adult-born neurons in the dentate gyrus of the hippocampus. Consistent with enhanced context discrimination in aged DN-p38αAF/+, we discovered enhanced production of adult-born neurons in the dentate gyrus of DN-p38αAF/+ mice compared to wildtype littermates. Our findings support the notion that p38α inhibition has therapeutic utility in aging diseases that affect cognition, such as AD.

p38MAPK builds a hyaluronan cancer niche to drive lung tumorigenesis.

Expansion of neoplastic lesions generates the initial signal that instigates the creation of a tumor niche. Nontransformed cell types within the microenvironment continuously coevolve with tumor cells to promote tumorigenesis. Here, we identify p38MAPK as a key component of human lung cancer, and specifically stromal interactomes, which provides an early, protumorigenic signal in the tissue microenvironment. We found that lung cancer growth depends on short-distance cues produced by the cancer niche in a p38-dependent manner. We identified fibroblast-specific hyaluronan synthesis at the center of p38-driven tumorigenesis, which regulates early stromal fibroblast activation, the conversion to carcinoma-associated fibroblasts (CAFs), and cancer cell proliferation. Systemic down-regulation of p38MAPK signaling in a knock-in model with substitution of activating Tyr182 to phenylalanine or conditional ablation of p38 in fibroblasts has a significant tumor-suppressive effect on K-ras lung tumorigenesis. Furthermore, both Kras-driven mouse lung tumors and orthotopically grown primary human lung cancers show a significant sensitivity to both a chemical p38 inhibitor and an over-the-counter inhibitor of hyaluronan synthesis. We propose that p38MAPK-hyaluronan-dependent reprogramming of the tumor microenvironment plays a critical role in driving lung tumorigenesis, while blocking this process could have far-reaching therapeutic implications.

Attenuation of p38α MAPK stress response signaling delays the in vivo aging of skeletal muscle myofibers and progenitor cells.

Functional competence and self-renewal of mammalian skeletal muscle myofibers and progenitor cells declines with age. Progression of the muscle aging phenotype involves the decline of juvenile protective factorsi.e., proteins whose beneficial functions translate directly to the quality of life, and self-renewal of progenitor cells. These characteristics occur simultaneously with the age-associated increase of p38α stress response signaling. This suggests that the maintenance of low levels of p38α activity of juvenile tissues may delay or attenuate aging. We used the dominant negative haploinsufficient p38α mouse (DN-p38α(AF/+)) to demonstrate that in vivo attenuation of p38α activity in the gastrocnemius of the aged mutant delays age-associated processes that include: a) the decline of the juvenile protective factors, BubR1, aldehyde dehydrogenase 1A (ALDH1A1), and aldehyde dehydrogenase 2 (ALDH2); b) attenuated expression of p16(Ink4a) and p19(Arf) tumor suppressor genes of the Cdkn2a locus; c) decreased levels of hydroxynonenal protein adducts, expression of COX2 and iNOS; d) decline of the senescent progenitor cell pool level and d) the loss of gastrocnemius muscle mass. We propose that elevated P-p38α activity promotes skeletal muscle aging and that the homeostasis of p38α impacts the maintenance of a beneficial healthspan.

Oncogene-mediated regulation of p53 ISGylation and functions.

Oncogene-mediated cellular transformation is a multistep process involving activation of growth-promoting pathways as well as inactivation of tumor suppressors. We recently found that ISGylation of the p53 tumor suppressor is an important novel mechanism to control its stability. Here we identified that Isg15-dependent regulation of p53 can be enhanced by different oncogenes. We further show that the Src-mediated phosphorylation of p53 on Tyr126 and Tyr220 has a positive effect on p53 ISGylation by enhancing Herc5 binding. In turn, deletion of Isg15 results in accumulation and activation of native p53 in transformed cells thus increasing its anti-cancer activity and suppressing tumorigenesis in mice. We propose that Isg15-dependent degradation of p53 is an alternative pathway for oncogenes to regulate p53 activity, and thus is an attractive pathway for development of new anti-cancer drugs.

Phosphatase WIP1 regulates adult neurogenesis and WNT signaling during aging.

The number of newly formed neurons declines rapidly during aging, and this decrease in neurogenesis is associated with decreased function of neural stem/progenitor cells (NPCs). Here, we determined that a WIP1-dependent pathway regulates NPC differentiation and contributes to the age-associated decline of neurogenesis. Specifically, we found that WIP1 is expressed in NPCs of the mouse subventricular zone (SVZ) and aged animals with genetically enhanced WIP1 expression exhibited higher NPC numbers and neuronal differentiation compared with aged WT animals. Additionally, augmenting WIP1 expression in aged animals markedly improved neuron formation and rescued a functional defect in fine odor discrimination in aged mice. We identified the WNT signaling pathway inhibitor DKK3 as a key downstream target of WIP1 and found that expression of DKK3 in the SVZ is restricted to NPCs. Using murine reporter strains, we determined that DKK3 inhibits neuroblast formation by suppressing WNT signaling and Dkk3 deletion or pharmacological activation of the WNT pathway improved neuron formation and olfactory function in aged mice. We propose that WIP1 controls DKK3-dependent inhibition of neuronal differentiation during aging and suggest that regulating WIP1 levels could prevent certain aspects of functional decline of the aging brain.

Isg15 controls p53 stability and functions.

Degradation of p53 is a cornerstone in the control of its functions as a tumor suppressor. This process is attributed to ubiquitin-dependent modification of p53. In addition to polyubiquitination, we found that p53 is targeted for degradation through ISGylation. Isg15, a ubiquitin-like protein, covalently modifies p53 at 2 sites in the N and C terminus, and ISGylated p53 can be degraded by the 20S proteasome. ISGylation primarily targets a misfolded, dominant-negative p53, and Isg15 deletion in normal cells results in suppression of p53 activity and functions. We propose that Isg15-dependent degradation of p53 represents an alternative mechanism of controlling p53 protein levels, and, thus, it is an attractive pathway for drug discovery.

Wip1 controls global heterochromatin silencing via ATM/BRCA1-dependent DNA methylation.

Wip1 phosphatase is emerging as an important regulator of tumorigenesis, but no unifying mechanistic network has been proposed. We found that Wip1 plays a key role in the transcriptional regulation of heterochromatin-associated DNA sequences. Wip1 was required for epigenetic remodeling of repetitive DNA elements through regulation of BRCA1 interaction with HP1, the recruitment of DNA methyltransferases, and subsequent DNA methylation. Attenuation of ATM, in turn, reversed heterochromatin methylation. This mechanism was critical for the recruitment of the AID cytidine deaminase, and Wip1 levels strongly correlated with C-to-T substitutions and a total mutation load in primary breast cancers. We propose that Wip1 plays an important role in the regulation of global heterochromatin silencing and thus is critical in maintaining genome integrity.

Genetic variants and mutations of PPM1D control the response to DNA damage.

The Wip1 phosphatase is an oncogene that is overexpressed in a variety of primary human cancers. We were interested in identifying genetic variants that could change Wip1 activity. We identified 3 missense SNPs of the human Wip1 phosphatase, L120F, P322Q, and I496V confer a dominant-negative phenotype. On the other hand, in primary human cancers, PPM1D mutations commonly result in a gain-of-function phenotype, leading us to identify a hot-spot truncating mutation at position 525. Surprisingly, we also found a significant number of loss-of-function mutations of PPM1D in primary human cancers, both in the phosphatase domain and in the C terminus. Thus, PPM1D has evolved to generate genetic variants with lower activity, potentially providing a better fitness for the organism through suppression of multiple diseases. In cancer, however, the situation is more complex, and the presence of both activating and inhibiting mutations requires further investigation to understand their contribution to tumorigenesis.

Apoptosis differently affects lineage tracing of Lgr5 and Bmi1 intestinal stem cell populations.

Emerging lineage-tracing data support the existence of several pools of intestinal stem cells (ISCs) in the adult mouse. The +4 location is known to harbor proliferative cells undergoing robust apoptosis in response to irradiation, but their relationship with recently reported ISC models is unclear. Here, we found that tamoxifen, at doses commonly used to induce lineage tracing, mimics the irradiation-induced apoptotic response of the +4 cells. We found that about 40% of apoptotic cells were Lgr5-positive whereas Bmi1-positive ISCs became sensitive to tamoxifen upon entering a proliferative state. In turn, when we suppressed apoptosis by either Bcl2 overexpression or Chk2 deletion, we found that lineage tracing of Lgr5-positive cells was efficiently reduced. In contrast, lineage tracing from Bmi1-positive ISCs was substantially increased in apoptosis-deficient backgrounds. We propose that apoptosis plays an important role in controlling lineage tracing from different ISC populations in the mouse intestine.

Hormone-sensing cells require Wip1 for paracrine stimulation in normal and premalignant mammary epithelium.

INTRODUCTION: The molecular circuitry of different cell types dictates their normal function as well as their response to oncogene activation. For instance, mice lacking the Wip1 phosphatase (also known as PPM1D; protein phosphatase magnesium-dependent 1D) have a delay in HER2/neu (human epidermal growth factor 2), but not Wnt1-induced mammary tumor formation. This suggests a cell type-specific reliance on Wip1 for tumorigenesis, because alveolar progenitor cells are the likely target for transformation in the MMTV(mouse mammary tumor virus)-neu but not MMTV-wnt1 breast cancer model. METHODS: In this study, we used the Wip1-knockout mouse to identify the cell types that are dependent on Wip1 expression and therefore may be involved in the early stages of HER2/neu-induced tumorigenesis. RESULTS: We found that alveolar development during pregnancy was reduced in Wip1-knockout mice; however, this was not attributable to changes in alveolar cells themselves. Unexpectedly, Wip1 allows steroid hormone-receptor-positive cells but not alveolar progenitors to activate STAT5 (signal transducer and activator of transcription 5) in the virgin state. In the absence of Wip1, hormone-receptor-positive cells have significantly reduced transcription of RANKL (receptor activator of nuclear factor kappa-B ligand) and IGF2 (insulin-like growth factor 2), paracrine stimulators of alveolar development. In the MMTV-neu model, HER2/neu activates STAT5 in alveolar progenitor cells independent of Wip1, but HER2/neu does not override the defect in STAT5 activation in Wip1-deficient hormone-sensing cells, and paracrine stimulation remains attenuated. Moreover, ERK (extracellular signal-regulated kinase) activation by HER2/neu in hormone-sensing cells is also Wip1 dependent. CONCLUSIONS: We identified Wip1 as a potentiator of prolactin and HER2/neu signaling strictly in the molecular context of hormone-sensing cells. Furthermore, our findings highlight that hormone-sensing cells convert not only estrogen and progesterone but also prolactin signals into paracrine instructions for mammary gland development. The instructive role of hormone-sensing cells in premalignant development suggests targeting Wip1 or prolactin signaling as an orthogonal strategy for inhibiting breast cancer development or relapse.