Christopher K. Patil

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion

Cellular senescence suppresses cancer by stably arresting the proliferation of damaged cells. Paradoxically, senescent cells also secrete factors that alter tissue microenvironments. The pathways regulating this secretion are unknown. We show that damaged human cells develop persistent chromatin lesions bearing hallmarks of DNA double-strand breaks (DSBs), which initiate increased secretion of inflammatory cytokines such as interleukin-6 (IL-6). Cytokine secretion occurred only after establishment of persistent DNA damage signalling, usually associated with senescence, not after transient DNA damage responses (DDRs). Initiation and maintenance of this cytokine response required the DDR proteins ATM, NBS1 and CHK2, but not the cell-cycle arrest enforcers p53 and pRb. ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence. Furthermore, DDR activity and IL-6 were elevated in human cancers, and ATM-depletion suppressed the ability of senescent cells to stimulate IL-6-dependent cancer cell invasiveness. Thus, in addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.

p38MAPK is a novel DNA damage response‐independent regulator of the senescence‐associated secretory phenotype

Cellular senescence suppresses cancer by forcing potentially oncogenic cells into a permanent cell cycle arrest. Senescent cells also secrete growth factors, proteases, and inflammatory cytokines, termed the senescence‐associated secretory phenotype (SASP). Much is known about pathways that regulate the senescence growth arrest, but far less is known about pathways that regulate the SASP. We previously showed that DNA damage response (DDR) signalling is essential, but not sufficient, for the SASP, which is restrained by p53. Here, we delineate another crucial SASP regulatory pathway and its relationship to the DDR and p53. We show that diverse senescence‐inducing stimuli activate the stress‐inducible kinase p38MAPK in normal human fibroblasts. p38MAPK inhibition markedly reduced the secretion of most SASP factors, constitutive p38MAPK activation was sufficient to induce an SASP, and p53 restrained p38MAPK activation. Further, p38MAPK regulated the SASP independently of the canonical DDR. Mechanistically, p38MAPK induced the SASP largely by increasing NF‐κB transcriptional activity. These findings assign p38MAPK a novel role in SASP regulation—one that is necessary, sufficient, and independent of previously described pathways.

A Human-Like Senescence-Associated Secretory Phenotype Is Conserved in Mouse Cells Dependent on Physiological Oxygen

Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP), which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that “senescent” mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen), do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3%) oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-α. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP.

Tumor Suppressor and Aging Biomarker p16INK4a Induces Cellular Senescence without the Associated Inflammatory Secretory Phenotype

Cellular senescence suppresses cancer by preventing the proliferation of cells that experience potentially oncogenic stimuli. Senescent cells often express p16INK4a, a cyclin-dependent kinase inhibitor, tumor suppressor, and biomarker of aging, which renders the senescence growth arrest irreversible. Senescent cells also acquire a complex phenotype that includes the secretion of many cytokines, growth factors, and proteases, termed a senescence-associated secretory phenotype (SASP). The SASP is proposed to underlie age-related pathologies, including, ironically, late life cancer. Here, we show that ectopic expression of p16INK4a and another cyclin-dependent kinase inhibitor, p21CIP1/WAF1, induces senescence without a SASP, even though they induced other features of senescence, including a stable growth arrest. Additionally, human fibroblasts induced to senesce by ionizing radiation or oncogenic RAS developed a SASP regardless of whether they expressed p16INK4a. Cells induced to senesce by ectopic p16INK4a expression lacked paracrine activity on epithelial cells, consistent with the absence of a functional SASP. Nonetheless, expression of p16INK4a by cells undergoing replicative senescence limited the accumulation of DNA damage and premature cytokine secretion, suggesting an indirect role for p16INK4a in suppressing the SASP. These findings suggest that p16INK4a-positive cells may not always harbor a SASP in vivo and, furthermore, that the SASP is not a consequence of p16INK4a activation or senescence per se, but rather is a damage response that is separable from the growth arrest.