Francis Rodier

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.

A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells

The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.

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.

p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes

In addition to allowing immune cells to signal tissue damage, HMBG1 is secreted by senescent cells to initiate inflammatory cytokine secretion.

Ionizing radiation‐induced long‐term expression of senescence markers in mice is independent of p53 and immune status

Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR‐induced senescence markers could persist long‐term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45u2003weeks, the maximum time analyzed) DNA damage foci and increased p16INK4a expression, two hallmarks of cellular senescence and aging. BrdU‐labeling experiments revealed that IR‐induced damaged cells are preferentially eliminated, at least partially, in a tissue‐dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild‐type and Rag2−/−u2003γC−/− mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long‐term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.

Telomere dysfunction and cell survival: roles for distinct TIN2-containing complexes

Telomeres are maintained by three DNA-binding proteins (telomeric repeat binding factor 1 [TRF1], TRF2, and protector of telomeres 1 [POT1]) and several associated factors. One factor, TRF1-interacting protein 2 (TIN2), binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether subcomplexes also exist in vivo. We provide evidence for two TIN2 subcomplexes with distinct functions in human cells. We isolated these two TIN2 subcomplexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13 and TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist and that TIN2-15C–sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.

Mitochondrial DNA damage induces apoptosis in senescent cells

Senescence is a cellular response to damage and stress. The senescence response prevents cancer by suppressing the proliferation of cells with a compromised genome and contributes to optimal wound healing in normal tissues. Persistent senescent cells are also thought to drive aging and age-associated pathologies through their secretion of inflammatory factors that modify the tissue microenvironment and alter the function of nearby normal or transformed cells. Understanding how senescent cells alter the microenvironment would be aided by the ability to induce or eliminate senescent cells at will in vivo. Here, we combine the use of the synthetic nucleoside analog ganciclovir (GCV) with herpes simplex virus thymidine kinase (HSVtk) activity to create or eliminate senescent human cells. We show that low concentrations of GCV induce senescence through the accumulation of nuclear DNA damage while higher concentrations of GCV, similar to those used in vivo, kill non-dividing senescent cells via mitochondrial DNA (mtDNA) damage and caspase-dependent apoptosis. Using this system, we effectively eliminated xenografted normal human senescent fibroblasts or induced senescence in human breast cancer cells in vivo. Thus, cellular senescence and mtDNA damage are outcomes of synthetic nucleoside analog treatment, indicating that the GCV–HSVtk combination can be used effectively to promote the targeted formation or eradication of senescent cells.

p16(INK4a) -mediated suppression of telomerase in normal and malignant human breast cells.

The cyclin‐dependent kinase inhibitor p16INK4a (CDKN2A) is an important tumor suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti‐oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short‐term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self‐renewal.

Caspase-independent cytochrome c release is a sensitive measure of low-level apoptosis in cell culture models

Age‐associated loss of tissue function and several chronic diseases may derive in part from the cumulative effects of subtle changes in the level of apoptotic cell death. Because apoptosis is rapid and undetectable once complete, small changes in its incidence are difficult to detect, even in well‐controlled cell cultures. We describe a new apoptosis assay that provides greater sensitivity than conventional assays because it measures the accumulation of apoptotic cells. Human and mouse fibroblasts and human mammary epithelial cells that initiated apoptosis were preserved for 3 days by inhibiting caspase activity using the chemical inhibitor Q‐VD‐OPH (QVD). Cells suspended in the process of apoptosis were scored by immunostaining for cytochrome c, which redistributed from mitochondria in healthy cells to the cytoplasm in dying cells. This caspase‐independent cytochrome c release (CICR) assay was more sensitive than several conventional assays when apoptosis was induced by actinomycin D, and detected cumulative background levels of apoptosis over a 3‐day interval. Using this assay, we show that normal fibroblasts undergo very little apoptosis upon X‐irradiation, indicating dominance of the senescence response in this cell type. Further, apoptosis increased subtly but measurably when human mammary epithelial and skin fibroblast cells entered crisis, indicating that cell death during crisis is largely non‐apoptotic.

ATM suppresses SATB1-induced malignant progression in breast epithelial cells.

SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2), but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.