PLoS BIOLOGY Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor Jean-Philippe Coppe 1 , Christopher K. Patil 1[ , Francis Rodier 1,2[ , Yu Sun 3 , Denise P. Mun oz 1,2 , Joshua Goldstein 1¤ , Peter S. Nelson 3 , Pierre-Yves Desprez 1,4 , Judith Campisi 1,2* 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 2 Buck Institute for Age Research, Novato, California, United States of America, 3 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 California Pacific Medical Center Research Institute, San Francisco, California, United States of America 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. Citation: Coppe JP, Patil CK, Rodier F, Sun Y, Mun oz DP, et al. (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12): e301. doi:10.1371/journal.pbio.0060301 Introduction Cancer is a multistep disease in which cells acquire increasingly malignant phenotypes. These phenotypes are acquired in part by somatic mutations, which derange normal controls over cell proliferation (growth), survival, invasion, and other processes important for malignant tumorigenesis [1]. In addition, there is increasing evidence that the tissue microenvironment is an important determinant of whether and how malignancies develop [2,3]. Normal tissue environ- ments tend to suppress malignant phenotypes, whereas abnormal tissue environments such at those caused by inﬂammation can promote cancer progression. Cancer development is restrained by a variety of tumor suppressor genes. Some of these genes permanently arrest the growth of cells at risk for neoplastic transformation, a process termed cellular senescence [4–6]. Two tumor suppressor pathways, controlled by the p53 and p16INK4a/pRB proteins, regulate senescence responses. Both pathways integrate multiple aspects of cellular physiology and direct cell fate towards survival, death, proliferation, or growth arrest, depending on the context [7,8]. Several lines of evidence indicate that cellular senescence is a potent tumor-suppressive mechanism [4,9,10]. Many poten- tially oncogenic stimuli (e.g., dysfunctional telomeres, DNA PLoS Biology | www.plosbiology.org damage, and certain oncogenes) induce senescence [6,11]. Moreover, mutations that dampen the p53 or p16INK4a/pRB pathways confer resistance to senescence and greatly increase cancer risk [12,13]. Most cancers harbor mutations in one or both of these pathways [14,15]. Lastly, in mice and humans, a senescence response to strong mitogenic signals, such as those delivered by certain oncogenes, prevents premalignant lesions from progressing to malignant cancers [16–19]. Academic Editor: Julian Downward, Cancer Research UK, United Kingdom Received June 27, 2008; Accepted October 22, 2008; Published December 2, 2008 Copyright: O 2008 Coppe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abbreviations: CM, conditioned medium; DDR, DNA damage response; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial–mesenchymal transition; GSE, genetic suppressor element; IL, interleukin; MIT, mitoxantrone; PRE, presenescent; PrEC, normal human prostate epithelial cell; REP, replicative exhaustion; SASP, senescence-associated secretory phenotype; SEN, senescent; shRNA, short hairpin RNA; XRA, X-irradiation * To whom correspondence should be addressed. E-mail: jcampisi@lbl.gov [ These authors contributed equally to this work. ¤ Current address: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America December 2008 | Volume 6 | Issue 12 | e301

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

The JAK-STAT pathway at twenty.

OBJECTIVE To establish an indirect immunofluorescent test so as to improve the sensitivity and specificity of examination of antibodies to beta(2)-glycoprotein METHODS Full-length beta(2)GP cDNA was obtained from human hepatocellular cancer cell line HepG2 by RT-PCR and cloned into the mammalian expression vector pEGFP-C1 The recombinant plasmid pEGFP-beta(2)GP was transfected into HEp-2 cells RT-PCR, immunoblotting (IBT), confocal fluorescence microscopy, and indirect immunofluorescent test (IIF) were used to confirm the expression, localization, and antigenicity of fusion protein of green fluorescent protein (GFP) Serum specimens from 19 patients suspected as with secondary antiphospholipid syndrome (APS), 1 patient diagnosed as with primary APS, and 10 normal persons were detected with IIF-IgG-beta(2)GP1, ELISA-IgG-ACL, and ELISA-IgG-beta(2)GP I simultaneously RESULTS (1) The HEp-beta(2)GP I cells thus obtained retained their ability of expression of beta(2)GP-I-GFP for more than ten generations This beta(2)GP-I-GFP showed the antigenicity of beta(2)GP-I with a characteristic feature (2) Seven of the 20 serum specimens from APS patients showed characteristic immunofluorescent pattern No serum specimen from normal persons showed immunofluorescent staining The comparison of results of the three methods showed that the concordance between IIF-IgG-beta(2)GP I and ELISA-IgG-beta(2)GP I was the most perfect (Kappa = 0886) (3) HEp-beta(2)GP I retained the immunofluorescent property of HEp-2 cell CONCLUSION As a new kind of substrate of IIF, beta(2)GP I transfectant can be used to detect anti-beta(2)GP-I antibodies Transfeted HEp-2 cells keep the immunofluorescent property of HEp-2 cells in IFANA test and can be used as substrate for routine IFANA detection

[Expression of fusion proteins in beta(2)GP I gene-transfected HEp-2 cells and its clinical application].

Recent research has led to a better but as yet incomplete understanding of the complex roles osteopontin plays in mammalian physiology. A soluble protein found in all body fluids, it stimulates signal transduction pathways (via integrins and CD44 variants) similar to those stimulated by components of the extracellular matrix. This appears to promote the survival of cells exposed to potentially lethal insults such as ischemia/reperfusion or physical/chemical trauma. OPN is chemotactic for many cell types including macrophages, dendritic cells, and T cells; it enhances B lymphocyte immunoglobulin production and proliferation. In inflammatory situations it stimulates both pro- and anti-inflammatory processes, which on balance can be either beneficial or harmful depending on what other inputs the cell is receiving. OPN influences cell-mediated immunity and has been shown to have Th1-cytokine functions. OPN deficiency is linked to a reduced Th1 immune response in infectious diseases, autoimmunity and delayed type hypersensitivity. OPN's role in the central nervous system and in stress responses has also emerged as an important aspect related to its cytoprotective and immune functions. Evidence suggests that either OPN or anti-OPN monoclonal antibodies (depending on the circumstances) might be clinically useful in modulating OPN function. Manipulation of plasma OPN levels may be useful in the treatment of autoimmune disease, cancer metastasis, osteoporosis and some forms of stress.

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Osteopontin: Role in immune regulation and stress responses

The Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway was discovered 20 years ago as a mediator of cytokine signaling. Since this time, more than 2,500 articles have been published demonstrating the importance of this pathway in virtually all malignancies. Although there are dozens of cytokines and cytokine receptors, four Jaks, and seven Stats, it seems that interleukin-6–mediated activation of Stat3 is a principal pathway implicated in promoting tumorigenesis. This transcription factor regulates the expression of numerous critical mediators of tumor formation and metastatic progression. This review will examine the relative importance and function of this pathway in nonmalignant conditions as well as malignancies (including tumor intrinsic and extrinsic), the influence of other Stats, the development of inhibitors to this pathway, and the potential role of inhibitors in controlling or eradicating cancers.

Targeting the interleukin-6/Jak/stat pathway in human malignancies.

Expression of the type I transmembrane glycoprotein CD44 has recently been recognized as a signature for cancer stem cells. In this study, we demonstrate that CD44, once engaged, is internalized and translocated to the nucleus, where it binds to various promoters, including that of cyclin D1, leading to cell fate change through transcriptional reprogramming. In regulating cyclin D1 expression, the internalized CD44 forms a complex with STAT3 and p300 (acetyltransferase), eliciting STAT3 acetylation at lysine 685 and dimer formation in a cytokine- and growth factor–independent manner. A bipartite nuclear localization signal (NLS) was mapped to the cytoplasmic tail of CD44, which mediates its nuclear translocation. Expression of CD44(NLS) mutant sequesters STAT3 in cytosol. In the nucleus, the acetylated STAT3 dimer remains associated with CD44 and binds to the cyclin D1 promoter, leading to increased cyclin D1 expression and cell proliferation. This study describes a novel function for CD44 in transcriptional modulation through nuclear translocation of the internalized CD44 and complex formation with transcription factors.

/pdf/acetylation-and-activation-of-stat3-mediated-by-nuclear-1pu88fvici.pdf

Acetylation and activation of STAT3 mediated by nuclear translocation of CD44

Osteopontin (OPN) and splice variants of CD44 (CD44(V)) have independently been identified as markers for tumor progression. In this study, we show that both OPN and CD44(V) are frequently overexpressed in human gastric cancer and that OPN-engaged CD44(V) ligation confers cells an increased survival mediated through integrin activation. First, we show that OPN treatment confers cells an increased resistance to UV-induced apoptosis. The OPN-mediated antiapoptosis is dependent on the expression of the variant exon 6 (V6)- or V7-containing CD44 as shown by overexpression of individual CD44(V) in gastric AZ521 cells that express no or very low level of endogenous CD44 and by knockdown of the constitutively expressed V6-containing CD44 isoforms in colon HT29 cells. Although OPN also interacts with RGD integrins, OPN-RGD sequence is dispensable for OPN-mediated antiapoptosis. OPN-induced antiapoptosis is mainly attributed to the engagement of CD44(V) isoforms and the relay of an inside-out signaling via Src activity, leading to robust integrin activation. Furthermore, OPN-elicited antiapoptosis was observed when cells were plated on fibronectin but not on poly-D-lysin, and preincubation of cells with anti-integrin beta(1) antibody to block integrin-extracellular matrix (ECM) interaction or ectopic expression of the dominant-negative forms of focal adhesion kinase to block ECM-derived signal abolished OPN-induced survival, suggesting that OPN-elicited antiapoptotic function is propagated from matrix transduced by integrin. Taken together, we showed that OPN-CD44(V) interaction promotes ECM-derived survival signal mediated through integrin activation, which may play an important role in the pathogenic development and progression of gastric cancer.

https://cancerres.aacrjournals.org/content/canres/67/5/2089.full.pdf

Osteopontin Promotes Integrin Activation through Outside-In and Inside-Out Mechanisms: OPN-CD44V Interaction Enhances Survival in Gastrointestinal Cancer Cells

CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin β1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.

CD44 engagement promotes matrix-derived survival through the CD44-SRC-integrin axis in lipid rafts.

Rhotekin (RTKN), the gene coding for the Rho effector, RTKN, was shown to be overexpressed in human gastric cancer (GC). In this study, we further showed that RTKN is expressed at a low level in normal cells and is overexpressed in many cancer-derived cell lines. The function of RTKN as an effector protein in Rho GTPase-mediated pathways regulating apoptosis was investigated. By transfection and expression of RTKN in cells that expressed endogenous RTKN at a low basal level, we showed that RTKN overexpression conferred cell resistance to apoptosis induced by serum deprivation or treatment with sodium butyrate, and the increased resistance correlated to the level of RTKN. Conversely, reducing RTKN expression by small interfering RNAs greatly sensitized cells to apoptosis. The RTKN-mediated antiapoptotic effect was blocked by the nuclear factor-kappaB (NF-kappaB) inhibitors, curcumin or parthenolide, but not by the phosphatidylinositol 3'-OH-kinase inhibitor, LY294002, or the MAP kinase inhibitor, PD98059. Reporter gene assays and electrophoretic mobility shift assay confirmed that RTKN overexpression led to constitutive activation of NF-kappaB through the phosphorylation of IkappaB by IKKbeta. By using the RTKN truncation mutants, we showed that RTKN mediated Rho activity eliciting signaling pathway to activate NF-kappaB, with a concomitant induction of expression of the NF-kappaB antiapoptotic genes, cIAP-2, BCl-xL, A1, and A20. Consistent with these data, RTKN-expressing cells showed increased chemoresistance to 5-fluorouracil and paclitaxol, and the resistance was greatly attenuated by NF-kappaB inhibitor. In conclusion, overactivated Rho/RTKN/NF-kappaB signaling pathway through overexpression of RTKN may play a key role in gastric tumorigenesis by conferring cells resistance to apoptosis, and this signaling pathway may serve as an important target for novel therapeutic approaches to the treatment of human GC.

Rho/Rhotekin-mediated NF-kappaB activation confers resistance to apoptosis

Serglycin (SRGN), previously recognized as an intracellular proteoglycan involved in the storage processes of secretory granules, has recently been shown to be upregulated in several solid tumors. We have previously shown that SRGN in non-small cell lung cancer (NSCLC) promotes malignant phenotypes in a CD44-dependent manner and increased expression of SRGN predicts poor prognosis of primary lung adenocarcinomas. However, the underlying mechanism remains to be defined. Overexpression, knockdown and knockout approaches were performed to assess the role of SRGN in cell motility using wound healing and Boyden chamber migration assays. SRGN devoid of glycosaminoglycan (GAG) modification was produced by site-directed mutagenesis or chondroitinase treatment. Liquid chromatography/tandem mass spectrometry was applied for quantitative analysis of the disaccharide compositions and sulfation extent of SRGN GAGs. Western blot and co-immunoprecipitation analyses were performed to determine the expression and interaction of proteins of interest. Actin cytoskeleton organization was monitored by immunofluorescence staining. SRGN expressed by NSCLC cells is readily secreted to the extracellular matrix in a heavily glycosylated form attached with mainly chondroitin sulfate (CS)-GAG chains, and to a lesser extent with heparin sulfate (HS). The CS-GAG moiety serves as the structural motif for SRGN binding to tumor cell surface CD44 and promotes cell migration. SRGN devoid of CS-GAG modification fails to interact with CD44 and has lost the ability to promote cell migration. SRGN/CD44 interaction promotes focal adhesion turnover via Src-mediated paxillin phosphorylation and disassembly of paxillin/FAK adhesion complex, facilitating cell migration. In support, depletion of Src activity or removal of CS-GAGs efficiently blocks SRGN-mediated Src activation and cell migration. SRGN also promotes cell migration via inducing cytoskeleton reorganization mediated through RAC1 and CDC42 activation accompanied with increased lamellipodia and filopodia formation. Proteoglycan SRGN promotes NSCLC cell migration via the binding of its GAG motif to CD44. SRGN/CD44 interaction induces Rho-family GTPase-mediated cytoskeleton reorganization and facilitates Src-mediated focal adhesion turnover, leading to increased cell migration. These findings suggest that targeting specific glycans in tumor microenvironment that serve as ligands for oncogenic pathways may be a potential strategy for cancer therapy.

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Mei-Jung Wang

Papers

Acetylation and activation of STAT3 mediated by nuclear translocation of CD44

Osteopontin Promotes Integrin Activation through Outside-In and Inside-Out Mechanisms: OPN-CD44V Interaction Enhances Survival in Gastrointestinal Cancer Cells

CD44 engagement promotes matrix-derived survival through the CD44-SRC-integrin axis in lipid rafts.

Rho/Rhotekin-mediated NF-kappaB activation confers resistance to apoptosis

Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44.