Stable Growth Arrest Research Articles

Cellular senescence as a target to inhibit epithelial‐mesenchymal transition in HK‐2 cells

Cellular senescence is a program that induces a stable growth arrest accompanied by metabolic reprogramming, phenotypic changes, increased senescence-associated β-galactosidase (SA-β-gal) activity, and secretion of senescence-associated secretory phenotype. It has been demonstrated that cellular senescence is associated with epithelial-mesenchymal transition (EMT) and chronic kidney disease, and higher SA-β-gal activity were found in proximal and distal tubules of feline chronic kidney disease (CKD). Studies showed that elimination of senescent cells by FOXO4 DRI, a peptide antagonist to block the interaction of forkhead box O 4 (FOXO4) and p53, improved kidney function in aging mice. The represent study tested the hypothesis that inhibition of cellular senescence alleviates TGF-β1-induced EMT in human renal epithelial (HK-2) cells. Cultured HK-2 cells were divided into 3 groups: Ctrl, TGF-β1 and FOXO4 DRI+TGF-β1. Western blot analysis showed that a senescent biomarker, p16, was significantly higher in TGF-β1 group than that in Ctrl and F+ TGF-β1 groups (1±0.15, 2±0.1, 0.96±0.15 in Ctrl, TGF-β1, F+ TGF-β1 group, respectively, p<0.05). Another important senescent indicator, SA-β-gal staining, was also significantly increased in TGF-β1 group than that in Ctrl and F+ TGF-β1 groups(1±0.27, 7.2±0.39, 4.6±0.38 in Ctrl, TGF-β1, F+ TGF-β1 group, respectively, p<0.05). Meanwhile, Western blot analysis showed that the EMT markers, α-SMA and collagen I/III, were significantly higher in TGF-β1 group than that in Ctrl and F+ TGF-β1 groups (α-SMA：1±0.05, 1.4±0.05, 0.96±0.1 in Ctrl, TGF-β1, F+ TGF-β1 group, respectively, p<0.05; and collagen I/III: 1±0.03, 1.49±0.06, 0.91±0.1 in Ctrl, TGF-β1, F+ TGF-β1 group, respectively, p<0.05). These results suggest that targeting cellular senescence might be a potential therapeutic strategy for CKD.

Abstract IA016: Cellular senescence in cancer therapy: Friend or foe?

Abstract Most used anti-cancer therapeutic approaches are based on impairing mitosis and targeting highly proliferative cells. The non-specificity of these interventions often leads to transient and chronic side effects, and a general accelerated aging phenotype. Cellular senescence - a complex stress response whereby cells lose irreversibly their capacity to proliferate – is potent tumor suppressive mechanism and a desired outcome of anti-cancer therapies. Our data show that chemotherapy agents induce senescent cells in both mice and humans. However, chemotherapy-induced senescent cells develop a strong secretory phenotype (SASP) and contribute to chemotoxicity in mice including bone marrow suppression, cardiac decline, decreased physical activity and cancer relapse. Interestingly, chemotherapy treatment combined to genetic or pharmacological senolysis delays or prevents the onset of several pathologies. We also show that similar to genotoxic chemotherapy, inhibitors of Cyclin-Dependent Kinases (CDK)-4/6 induce a p53-mediated senescence program. Interestingly, using RNAseq, cytokine arrays and in-tissue analyses we demonstrate that CDKi-induced senescent cells are characterized by a strongly reduced SASP. CDKi-induced senescent cells fail to promote paracrine detrimental effects, and do not lead to adverse effects in mice. Moreover, transient treatment with CDK4/6i in combination with chemotherapy is sufficient to reduce SASP and improve healthspan. Together, our data suggest that modulating the senescence response is a potent strategy to limit the toxicity of ant-cancer treatments. This effect can be achieved either by eliminating detrimental senescent cells or by designing drugs that can promote stable growth arrest without strong paracrine effects. Importantly, the phenotypical characterization of these different senescent cells could predict toxicity and side effects associated to standard oncological drugs. Citation Format: Marco Demaria. Cellular senescence in cancer therapy: Friend or foe? [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA016.

Early growth response 2 (EGR2) is a novel regulator of the senescence programme.

Senescence, a state of stable growth arrest, plays an important role in ageing and age‐related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up‐regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down‐regulates p16 levels and increases the pool of p16− p21− ‘reversed’ cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.

CDK4/6 Inhibition Promotes a P53-Dependent Senescent State with Restricted Secretory Phenotype

Cellular senescence is a state of stable growth arrest that acts as a tumor suppressive mechanism and desired outcome of anti-cancer interventions. Because of systemic administration and lack of tumor specificity, treatment with many anti-cancer drugs can cause premature senescence of non-malignant cells. These therapy-induced senescent cells can have profound detrimental pro-tumorigenic and pro-disease functions via activation of a pro-inflammatory secretory phenotype (SASP). Inhibitors of cyclin-dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence-like program in cancer cells, but their effect on normal cells, particularly in vivo, remains poorly characterized. Here, we observe that, in culture and in mice, exposure to CDK4/6i leads normal cells into a p53-dependent state of premature senescence. We demonstrate that the CDK4/6i-induced senescent program engages a SASP enriched in direct p53 targets (PASP) but without a pro-inflammatory and NF-κB-associated secretory phenotype (NASP). We show that tumor suppressive doses of CDK4/6i cause a PASP but not a NASP in tissues and plasma from mice and breast cancer patients. We also show that because lacking a NASP, CDK4/6i-induced senescent cells do not acquire pro-tumorigenic properties, and do not promote severe adverse reactions, in particular asthenia, in vivo. Our data suggest that senescent cells with a PASP but without a NASP maintain beneficial properties for cancer therapy without developing detrimental functions, thus representing a potentially less toxic and better tolerated outcome for interventions.

Therapy of lymphoma by immune checkpoint inhibitors: the role of T cells, NK cells and cytokine-induced tumor senescence

BackgroundAlthough antibodies blocking immune checkpoints have already been approved for clinical cancer treatment, the mechanisms involved are not yet completely elucidated. Here we used a λ-MYC transgenic model of endogenously...

Cellular senescence as a potential mediator of COVID-19 severity in the elderly.

SARS‐CoV‐2 is a novel betacoronavirus which infects the lower respiratory tract and can cause coronavirus disease 2019 (COVID‐19), a complex respiratory distress syndrome. Epidemiological data show that COVID‐19 has a rising mortality particularly in individuals with advanced age. Identifying a functional association between SARS‐CoV‐2 infection and the process of biological aging may provide a tractable avenue for therapy to prevent acute and long‐term disease. Here, we discuss how cellular senescence—a state of stable growth arrest characterized by pro‐inflammatory and pro‐disease functions—can hypothetically be a contributor to COVID‐19 pathogenesis, and a potential pharmaceutical target to alleviate disease severity. First, we define why older COVID‐19 patients are more likely to accumulate high levels of cellular senescence. Second, we describe how senescent cells can contribute to an uncontrolled SARS‐CoV‐2‐mediated cytokine storm and an excessive inflammatory reaction during the early phase of the disease. Third, we discuss the various mechanisms by which senescent cells promote tissue damage leading to lung failure and multi‐tissue dysfunctions. Fourth, we argue that a high senescence burst might negatively impact on vaccine efficacy. Measuring the burst of cellular senescence could hypothetically serve as a predictor of COVID‐19 severity, and targeting senescence‐associated mechanisms prior and after SARS‐CoV‐2 infection might have the potential to limit a number of severe damages and to improve the efficacy of vaccinations.

Targeting Cardiac Stem Cell Senescence to Treat Cardiac Aging and Disease.

Adult stem/progenitor are a small population of cells that reside in tissue-specific niches and possess the potential to differentiate in all cell types of the organ in which they operate. Adult stem cells are implicated with the homeostasis, regeneration, and aging of all tissues. Tissue-specific adult stem cell senescence has emerged as an attractive theory for the decline in mammalian tissue and organ function during aging. Cardiac aging, in particular, manifests as functional tissue degeneration that leads to heart failure. Adult cardiac stem/progenitor cell (CSC) senescence has been accordingly associated with physiological and pathological processes encompassing both non-age and age-related decline in cardiac tissue repair and organ dysfunction and disease. Senescence is a highly active and dynamic cell process with a first classical hallmark represented by its replicative limit, which is the establishment of a stable growth arrest over time that is mainly secondary to DNA damage and reactive oxygen species (ROS) accumulation elicited by different intrinsic stimuli (like metabolism), as well as external stimuli and age. Replicative senescence is mainly executed by telomere shortening, the activation of the p53/p16INK4/Rb molecular pathways, and chromatin remodeling. In addition, senescent cells produce and secrete a complex mixture of molecules, commonly known as the senescence-associated secretory phenotype (SASP), that regulate most of their non-cell-autonomous effects. In this review, we discuss the molecular and cellular mechanisms regulating different characteristics of the senescence phenotype and their consequences for adult CSCs in particular. Because senescent cells contribute to the outcome of a variety of cardiac diseases, including age-related and unrelated cardiac diseases like diabetic cardiomyopathy and anthracycline cardiotoxicity, therapies that target senescent cell clearance are actively being explored. Moreover, the further understanding of the reversibility of the senescence phenotype will help to develop novel rational therapeutic strategies.

Galactose-modified duocarmycin prodrugs as senolytics.

Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with cancer, fibrosis and many age‐related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence‐associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal β‐galactosidase, and this has been exploited as a marker for senescence (senescence‐associated β‐galactosidase activity). Consequently, we hypothesized that galactose‐modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose‐modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal β‐galactosidase (GLB1)‐dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole‐body irradiation treatment of mice. Moreover, taking advantage of a mouse model of adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD prodrug selectively reduced the number of β‐catenin‐positive preneoplastic senescent cells. In summary, the above results make a case for testing the potential of galactose‐modified duocarmycin prodrugs to treat senescence‐related pathologies.

SENESCENCE-ASSOCIATED SECRETOME COMPONENTS AS BIOMARKERS FOR AGING

Cellular senescence is a state of stable growth arrest in response to stress, which is a fundamental process of biological aging. They secrete products, the Senescence-Associated Secretory Phenotype (SASP), which consists of inflammatory cytokines, chemokines, growth factors and matrix remodeling proteins. Senescent cells accumulate with advancing age and partial elimination of senescent cells can reverse age-related dysfunction and increase mean lifespan in mice. However, it is not clear whether components of the SASP can be measured in human plasma and serve as aging biomarkers. Here we generated a candidate panel of senescence markers based on a multiplexed bead-based assay of proteins secreted by senescent preadipocytes, endothelial cells, fibroblasts, myoblasts, preadipocytes, and epithelial cells compared to non-senescent controls. The SASP undoubtedly varies by cell type; however, we observed that multiple components of the SASP are conserved. We then assessed circulating SASP components in human plasma samples from Mayo Clinic Biobank participants (n=280, 20 male and 20 female per decade, age 20-90) using the same method. We confirmed that components of the SASP can be quantified in human plasma with the multiplexed bead-based assay and observed several SASP components robustly increase with chronological age in humans. Our study illustrates that senescence-associated secretome components are detectable in human plasma and could potentially serve as biomarkers of systemic aging and senescent cell burden.

077 Type I immunity induces a senescence-like growth arrest in malaria parasites

Type I immunity kills and contains plasmodium species in mammalians. However, malaria tends to recur, showing that the natural type I immune response strongly reduces the pathogens but fails to eradicate them. Analyzing the effect of type I immunity on plasmodium-infected hepatocytes, we confirmed that indeed IFN (interferon)-γ strongly reduces parasite number. Surprisingly, we also found that IFN-γ induces in addition a senescence-like growth arrest in the remaining plasmodium parasites. One day of treatment with IFN-γ caused a stable growth-arrest over 14 days in surviving Plasmodium (P.) falciparum liver stages. IFN-γ induced small non-proliferating plasmodium forms in human, monkey and mouse hepatocytes. Atovaquone treatment killed the large majority of the remaining parasites confirming that these small forms are alive. In vitro data showed that mammalian p21 can interact with parasite cyclin-dependent kinases (CDK) PFMRK, PFPK5 and PFPK6. Inhibition of these CDKs with Artemisinin severely impairs the growth of P.falciparum, without killing the parasites. Therefore, we asked whether IFN-γ and plasmodium parasites induce p21 in mammalian hepatocytes and whether mammalian p21 can arrest P.falciparum in vivo. Liver-infection with plasmodium parasites leads to an interferon-response and induced p21 but not p16 protein levels inside primary hepatocytes. The p21 protein was further enhanced by treatment with IFN-γ and tumor necrosis factor. Most importantly, p210/0 hepatocytes had a major defect in containing malaria parasites in vitro. In consequence, infection of p210/0 mice had a major defect in controlling the onset and the extent parasitemia. Hence, type I immunity did not only kill malaria parasites but induced a p21-dependent senescence-like growth arrest in the remaining plasmodium stages. This IFN-γ induced senescence-like growth arrest significantly contributed to the containment of the disease.

Ribosomal protein RPL22/eL22 regulates the cell cycle by acting as an inhibitor of the CDK4-cyclin D complex

ABSTRACTSenescence is a tumor suppressor program characterized by a stable growth arrest while maintaining cell viability. Senescence-associated ribogenesis defects (SARD) have been shown to regulate senescence through the ability of the ribosomal protein S14 (RPS14 or uS11) to bind and inhibit the cyclin-dependent kinase 4 (CDK4). Here we report another ribosomal protein that binds and inhibits CDK4 in senescent cells: L22 (RPL22 or eL22). Enforcing the expression of RPL22/eL22 is sufficient to induce an RB and p53-dependent cellular senescent phenotype in human fibroblasts. Mechanistically, RPL22/eL22 can interact with and inhibit CDK4-Cyclin D1 to decrease RB phosphorylation both in vitro and in cells. Briefly, we show that ribosome-free RPL22/eL22 causes a cell cycle arrest which could be relevant during situations of nucleolar stress such as cellular senescence or the response to cancer chemotherapy.

NAD+ metabolism governs the proinflammatory senescence-associated secretome.

Cellular senescence is a stable growth arrest that is implicated in tissue ageing and cancer. Senescent cells are characterized by an upregulation of proinflammatory cytokines, which is termed the senescence-associated secretory phenotype (SASP). NAD+ metabolism influences both tissue ageing and cancer. However, the role of NAD+ metabolism in regulating the SASP is poorly understood. Here we show that nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway, governs the proinflammatory SASP independent of senescence-associated growth arrest. NAMPT is regulated by HMGAs during senescence. The HMGAs/NAMPT/NAD+ signaling axis promotes the proinflammatory SASP through enhancing glycolysis and mitochondrial respiration. HMGAs/NAMPT promotes the proinflammatory SASP through NAD+-mediated suppression of AMPK kinase, which suppresses p53-mediated inhibition of p38MAPK to enhance NFκb activity. We conclude that NAD+ metabolism governs the proinflammatory SASP. Given the tumor-promoting effects of the proinflammatory SASP, our results suggest that anti-ageing dietary NAD+ augmentation should be administered with precision.

CELLULAR SENESCENCE AND CHRONOLOGICAL AGE IN VARIOUS HUMAN TISSUES: A SYSTEMATIC REVIEW AND META-ANALYSIS

Background: Cellular senescence, a stable growth arrest of cells, is increasingly recognized as a driver of the aging process and of age-related diseases. Our aim was to systemically summarize the literature on the association between senescence and chronological age in human tissue. Methods: We searched Pubmed, Web of Science and Embase for articles that reported on senescence markers dependent on age in any human tissue. Out of 4838 unique articles, 51 articles were included in a systematic review, and 45 of these were suitable for meta-analysis. Data was extracted on the origin of tissue, senescence markers, age and gender distribution of donors and correlation coefficients. The Z-scores, transformed from the correlation coefficients through Fisher’s method, were pooled under a random effects model using Comprehensive Meta-Analysis Software. Heterogeneity was reported with the I-squared test. Results: Overall, the correlation coefficient between senescence and age was 0.276 (95% CI 0.225–0.325), with differences between tissue types. Highest mean correlation coefficients was found for brain (0.701), followed by lung (0.670). Mean correlation coefficient were lower for other tissues: artery (0.199), blood (0.290), eye (0.100), kidney (0.235), prostate (0.263) and skin (0.287). The I2 value was 67.7%. Conclusion: Senescence is significantly associated with chronological age in all human tissues. The effect size of association varies widely between tissues.

Abstract SY26-03: Dual targeting of senescence and tumor immunity for cancer therapy

Abstract Senescence induction in cancer works as a potent weapon to eradicate tumorigenesis. Therapies that enhance senescence not only promote a stable cell growth arrest but also work as a strong stimulus for the activation of the tumor immune response. However, cancer cells bypass senescence by either acquiring new genetic alterations or by disrupting the tumor immune response. Here, I will discuss the identification of oncogenic pathways that bypass Pten-loss-induced cellular senescence, thereby promoting prostate cancer progression and metastasis. Next, I will present evidence demonstrating that both tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) control senescence induction in chemotherapy-treated prostate cancers and that immunotherapies that interfere with either MDSCs' recruitment or the polarization of TAMs promote senescence in advanced PTEN-deficient prostate cancer. Citation Format: Andrea Alimonti. Dual targeting of senescence and tumor immunity for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY26-03.

The Effects of the Senescent Preadipocyte Secretome on Skeletal Muscle Cells

Adipocytes accumulate within and around skeletal muscle with advancing age. Adipose progenitor cells, preadipocytes, are prone to cellular senescence, a state of stable growth arrest associated with the secretion of cytokines, chemokines and matrix remodeling proteins, collectively referred to as the senescence‐associated secretory phenotype (SASP). Considerable evidence implicates the SASP in age‐related tissue inflammation and degeneration; however, its impact on skeletal muscle is poorly understood. Herein, we examined the effects of conditioned media (CM) collected from healthy human (control CM) and senescent by irradiation (senescent CM) preadipocytes on human skeletal muscle cells (SkMCs). Compared to control CM, senescent CM had more than 10‐fold higher concentrations of IL‐6, IL‐1β, IL‐8, PAI‐1, MCP‐1 and TNFα as quantified by a multiplexed antibody‐based assay. Senescent CM increased expression of IL‐6 (5‐fold), IL‐1α (1.6‐fold), IL‐1β (3.5‐fold), MCP‐1 (2.5‐fold), PAI‐1 (1.7‐fold) and MMP‐3 (3‐fold) in differentiated SkMCs relative to control CM as measured by qPCR (all p &lt; 0.05). Senescent CM increased proliferation of undifferentiated SKMCs by 20%; however, inhibited differentiation relative to control CM per the fusion index. Preliminary data further suggest senescent CM compromises insulin‐ and IGF‐1‐mediated activation of Akt. Collectively, our data suggest that the SASP of senescent preadipocytes may drive inflammation in SkMCs and compromise their differentiation and, potentially, response to metabolic an anabolic stimuli. These effects may contribute to age‐related changes in skeletal muscle health and regeneration.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

E47 Governs the MYC-CDKN1B/p27KIP1-RB Network to Growth Arrest PDA Cells Independent of CDKN2A/p16INK4A and Wild-Type p53.

Background & AimsOncogenic mutations in KRAS, coupled with inactivation of p53, CDKN2A/p16INK4A, and SMAD4, drive progression of pancreatic ductal adenocarcinoma (PDA). Overexpression of MYC and deregulation of retinoblastoma (RB) further promote cell proliferation and make identifying a means to therapeutically alter cell-cycle control pathways in PDA a significant challenge. We previously showed that the basic helix-loop-helix transcription factor E47 induced stable growth arrest in PDA cells in vitro and in vivo. Here, we identified molecular mechanisms that underlie E47-induced growth arrest in low-passage, patient-derived primary and established PDA cell lines.MethodsRNA sequencing was used to profile E47-dependent transcriptomes in 5 PDA cell lines. Gene Ontology analysis identified cell-cycle control as the most altered pathway. Small interfering RNA/short hairpin RNA knockdown, small-molecule inhibitors, and viral expression were used to examine the function of E47-dependent genes in cell-cycle arrest. Cell morphology, expression of molecular markers, and senescence-associated β-galactosidase activity assays identified cellular senescence.ResultsE47 uniformly inhibited PDA cell-cycle progression by decreasing expression of MYC, increasing the level of CDKN1B/p27KIP1, and restoring RB tumor-suppressor function. The molecular mechanisms by which E47 elicited these changes included altering both RNA transcript levels and protein stability of MYC and CDKN1B/p27KIP1. At the cellular level, E47 elicited a senescence-like phenotype characterized by increased senescence-associated β-galactosidase activity and altered expression of senescence markers.ConclusionsE47 governs a highly conserved network of cell-cycle control genes, including MYC, CDKN1B/p27KIP1, and RB, which can induce a senescence-like program in PDA cells that lack CDKN2A/p16INK4A and wild-type p53. RNA sequencing data are available at the National Center for Biotechnology Information GEO at https://www.ncbi.nlm.nih.gov/geo/; accession number: GSE100327.

Sensing the Breaks: Cytosolic Chromatin in Senescence and Cancer

Cellular senescence constitutes a stable growth arrest characterized by DNA damage response (DDR) activation and by the senescence-associated secretory phenotype (SASP). SASP, through its paracrine effects, stimulates the immune system for senescence eradication. Similarly, chemotherapy-treated cancers activate an interferon-mediated response to induce anti-tumor immunity. Recent studies now uncover a new role for the innate DNA sensing pathway in the recognition of cytosolic chromatin in senescence and cancer.

Senescence and aging: Causes, consequences, and therapeutic avenues.

Aging is the major risk factor for cancer, cardiovascular disease, diabetes, and neurodegenerative disorders. Although we are far from understanding the biological basis of aging, research suggests that targeting the aging process itself could ameliorate many age-related pathologies. Senescence is a cellular response characterized by a stable growth arrest and other phenotypic alterations that include a proinflammatory secretome. Senescence plays roles in normal development, maintains tissue homeostasis, and limits tumor progression. However, senescence has also been implicated as a major cause of age-related disease. In this regard, recent experimental evidence has shown that the genetic or pharmacological ablation of senescent cells extends life span and improves health span. Here, we review the cellular and molecular links between cellular senescence and aging and discuss the novel therapeutic avenues that this connection opens.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated β-galactosidase (SA-β-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal β-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry(IHC). The major limitation of the SA-β-Gal assay is the requirement of fresh or frozen samples. Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-β-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we useIHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

A screen for inducers of bHLH activity identifies pitavastatin as a regulator of p21, Rb phosphorylation and E2F target gene expression in pancreatic cancer.

The average survival for patients with Pancreatic Ductal Adenocarcinoma (PDA) is merely 6 months, underscoring the need for new therapeutic approaches. During PDA progression, pancreatic acinar cells lose activity of the ClassI/II bHLH factors that regulate quiescence. We previously found that promoting transcriptional activity of the Class I bHLH factor E47 in highly aggressive PDA cells induced stable growth arrest in vitro and in vivo. To translate these findings for clinical utility, we developed a high throughput screening platform to identify small molecule inducers of Class I/II bHLH activity. A screen of 4,375 known drugs identified 70 bHLH activators. Prominent among the hits were members of the statin class of HMG-CoA reductase inhibitors, cholesterol lowering drugs that are also being evaluated in cancer. Studies with pitavastatin in primary patient derived tumor cells and established PDA lines, revealed dose dependent growth inhibition. At the molecular level, pitavastatin induced expression of the cyclin dependent kinase (CDK) inhibitor p21 in a cholesterol independent manner, blocked repressive phosphorylation of the Retinoblastoma tumor suppressor protein at CDK targeted sites, and reduced expression of E2F target genes required for progression through the G1/S boundary. Together, the data provide new insight into mechanisms by which statins constrain proliferation in cancer and establish the effectiveness of a novel screening platform to identify small molecules of clinical relevance in pancreatic cancer.