Coming of age: molecular drivers of aging and therapeutic opportunities

Aging is like the weather: everyone talks about it, but no one seems to do anything about it. We believe this may soon change, as an improved understanding of the molecular and genetic pathways underlying aging suggests it is possible to therapeutically target the aging process and increase health span. This Review series focuses on fundamental cellular mechanisms of aging and their relationship to human disease. These pathways include telomere dysfunction in cellular senescence and induction of the senescence-associated secretory phenotype (SASP) in systemic aging, sirtuin family regulation of metabolism and aging-associated diseases, mitochondrial metabolism in aging, the mechanistic target of rapamycin (mTOR) signaling pathway and the use of mTOR inhibitors to increase longevity, the progressive decline of the immune system with age, and aging-associated changes to pancreatic islet β cells that may contribute to diabetes. Together, these articles explore pathways affecting aging and possible interventional targets to slow or delay the onset of age-related pathologies

The Regulation of INK4/ARF in Cancer and Aging

Loss of the // locus on chromosome 9p21 is among the most frequent cytogenetic events in human cancer. The products of the locus—p15, p16, and ARF—play widespread and independent roles in tumor suppression. Recent data also suggest that expression of p16 induces an age-dependent decrease in the proliferative capacity of certain tissue-specific stem cells and unipotent progenitors. Here, we discuss the regulation and role of p16, ARF, and p15 in cancer and aging

Detecting and characterizing circular RNAs

Circular RNA transcripts were first identified in the early 1990s but knowledge of these species has remained limited, as their study has been difficult through traditional methods of RNA analysis. Now, novel bioinformatic approaches coupled with biochemical enrichment strategies and deep sequencing have allowed comprehensive studies of circular RNA species. Recent studies have revealed thousands of endogenous circular RNAs (circRNAs) in mammalian cells, some of which are highly abundant and evolutionarily conserved. Evidence is emerging that some circRNAs might regulate microRNA (miRNA) function, and roles in transcriptional control have also been suggested. Therefore, study of this class of non-coding RNAs has potential implications for therapeutic and research applications. We believe the key future challenge to the field will be to understand the regulation and function of these unusual molecules

Defining the toxicology of aging

Mammalian aging is complex and incompletely understood. While significant effort has been spent addressing the genetics or, more recently, the pharmacology of aging, the toxicology of aging has been relatively understudied. Just as an understanding of `carcinogens' has proven critical to modern cancer biology, an understanding of environmental toxicants that accelerate aging (`gerontogens') will inform gerontology. In this review, we discuss the evidence for the existence of mammalian gerontogens, as well as describe biomarkers needed to measure the age-promoting activity of a given toxicant. We focus on the effects of putative gerontogens on the in vivo accumulation of senescent cells, a characteristic feature of aging that plays a causal role in some age-associated phenotypes

Review: A Meta-Analysis of GWAS Studies and Age-Associated Diseases

Genome-Wide Association studies (GWAS) offer an unbiased means to understand the genetic basis of traits by identifying single nucleotide polymorphisms (SNPs) linked to causal variants of complex phenotypes. GWAS have identified a host of susceptibility SNPs associated with many important human diseases, including diseases associated with aging. In an effort to understand the genetics of broad resistance to age-associated diseases (i.e. ‘wellness’), we performed a meta-analysis of human GWAS. Toward that end, we compiled 372 GWAS that identified 1,775 susceptibility SNPs to 105 unique diseases and used these SNPs to create a genomic landscape of disease susceptibility. This map was constructed by partitioning the genome into 200 kb ‘bins’ and mapping the 1,775 susceptibility SNPs to bins based on their genomic location. Investigation of these data revealed significant heterogeneity of disease association within the genome, with 92% of bins devoid of disease-associated SNPs. In contrast, 10 bins (0.06%) were significantly (pINK4/ARF (CDKN2a/b) tumor suppressor locus on 9p21.3. Provocatively, all 10 significantly enriched bins contained genes linked to either inflammation or cellular senescence pathways, and SNPs near regulators of senescence were particularly associated with disease of aging (e.g. cancer, atherosclerosis, type 2 diabetes, glaucoma). This analysis suggests that germline genetic heterogeneity in the regulation of immunity and cellular senescence influences the human health span

Cells exhibiting strong p16INK4a promoter activation in vivo display features of senescence

The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by “knocking-in” a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the p16 INK4a locus. We used this allele (p16 tdTom ) for the enumeration, isolation, and characterization of individual p16 INK4a -expressing cells (tdTom + ). The half-life of the knocked-in transcript was shorter than that of the endogenous p16 INK4a mRNA, and therefore reporter expression better correlated with p16 INK4a promoter activation than p16 INK4a transcript abundance. The frequency of tdTom + cells increased with serial passage in cultured murine embryo fibroblasts from p16 tdTom/+ mice. In adult mice, tdTom + cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging. Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16 INK4a and found that tdTom + macrophages exhibited some features of senescence, including reduced proliferation, senescence-associated β-galactosidase (SA-β-gal) activation, and increased mRNA expression of a subset of transcripts encoding factors involved in SA-secretory phenotype (SASP). These results indicate that cells harboring activation of the p16 INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence

Metastasis in an orthotopic murine model of melanoma is independent of RAS/RAF mutation

Melanoma is the most lethal skin tumor, in large part because of a propensity for early metastasis. Good models of this most clinically relevant feature of melanoma are lacking. Here we report the development of an in vivo model of metastasis that relies on orthotopic injection of GFP-tagged lines in immunodeficient mice, serial intravital imaging of tumor progression and quantification of distant spread by 2-photon laser scanning microscopy, immunohistochemistry and real-time PCR analysis. Using this system, we report an assessment of the in vivo growth and metastatic properties of 11 well-characterized human melanoma cell lines. A subset of lines demonstrated rapid in vivo growth with invasion of host vasculature and distant seeding of viscera in this system. The ability to form metastasis in vivo did not correlate with 3D collagen invasion in vitro. Surprisingly, similar lines in terms of molecular genetic events differed markedly in their propensity to metastasize to distant organs such as brain and lung. In particular, two lines harboring B-RAF mutation and high levels of phosphorylated ERK and AKT (pERK and pAKT) were reproducibly unable to form tumors after orthotopic injection. Likewise, two previously identified RAS/RAF wild-type “epithelial-like” lines that do not have elevated pERK, pAKT or express TWIST1 mRNA still demonstrated a pronounced ability for orthotopic growth and metastatic spread. All the metastatic cell lines in this model showed increased NEDD9 expression, but NEDD9 lentiviral overexpression did not convey a metastatic phenotype on non-metastatic cells. These data suggest that melanoma metastasis is a molecularly heterogeneous process that may not require epidermal-to-mesenchymal transition or ERK activation, although both may facilitate the process

Identification of Human Papillomavirus Infection in Cancer Tissue by Targeted Next-generation Sequencing

Human papillomaviruses (HPV) are oncogenic DNA viruses implicated in squamous cell carcinomas of several anatomic sites, as well as endocervical adenocarcinomas. Identification of HPV is an actionable finding in some carcinomas, potentially influencing tumor classification, prognosis, and management. We incorporated capture probes for oncogenic HPV strains 16 and 18 into a broader next-generation sequencing (NGS) panel designed to identify actionable mutations in solid malignancies. A total of 21 head and neck, genitourinary and gynecological squamous cell carcinomas and endocervical adenocarcinomas were sequenced as part of the UNCSeq project. Using p16 immunohistochemical results as the gold standard, we set a cutoff for proportion of aligned HPV reads that maximized performance of our NGS assay (92% sensitive, 100% specific for HPV). These results suggest that sequencing of oncogenic pathogens can be incorporated into targeted NGS panels, extending the clinical utility of genomic assays

Chemotherapy and Stem Cell Transplantation Increase p16

AbstractThe expression of markers of cellular senescence increases exponentially in multiple tissues with aging. Age-related physiological changes may contribute to adverse outcomes in cancer survivors. To investigate the impact of high dose chemotherapy and stem cell transplantation on senescence markers in vivo, we collected blood and clinical data from a cohort of 63 patients undergoing hematopoietic cell transplantation. The expression of p16INK4a, a well-established senescence marker, was determined in T-cells before and 6months after transplant. RNA sequencing was performed on paired samples from 8 patients pre- and post-cancer therapy. In patients undergoing allogeneic transplant, higher pre-transplant p16INK4a expression was associated with a greater number of prior cycles of chemotherapy received (p=0.003), prior autologous transplantation (p=0.01) and prior exposure to alkylating agents (p=0.01). Transplantation was associated with a marked increase in p16INK4a expression 6months following transplantation. Patients receiving autologous transplant experienced a larger increase in p16INK4a expression (3.1-fold increase, p=0.002) than allogeneic transplant recipients (1.9-fold increase, p=0.0004). RNA sequencing of T-cells pre- and post- autologous transplant or cytotoxic chemotherapy demonstrated increased expression of transcripts associated with cellular senescence and physiological aging. Cytotoxic chemotherapy, especially alkylating agents, and stem cell transplantation strongly accelerate expression of a biomarker of molecular aging in T-cells