Surfaceome analysis of extracellular vesicles from senescent cells uncovers uptake repressor DPP4.

Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.

Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest.

Senescent cells release a variety of cytokines, proteases, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Sustained SASP contributes to a pattern of chronic inflammation associated with aging and implicated in many age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor; encoded by the TNFSF13B gene), a SASP protein, in multiple senescence models. We first characterized BAFF production across different senescence paradigms, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We then identified IRF1 (interferon regulatory factor 1) as a transcription factor required for promoting TNFSF13B mRNA transcription in senescence. We discovered that suppressing BAFF production decreased the senescent phenotype of both fibroblasts and monocyte-like cells, reducing IL6 secretion and SA-ß-Gal staining. Importantly, however, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-κB and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). We propose that BAFF is elevated across senescence models and is a potential target for senotherapy.

Ablation of cDC2 development by triple mutations within the Zeb2 enhancer.

The divergence of the common dendritic cell progenitor1-3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors-such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer4,6-but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the -165 kb Zeb2 enhancer8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPß. In vivo mutational analysis using CRISPR-Cas9 targeting showed that these NFIL3-C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3-C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (TH2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths9-11. Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the -165 kb Zeb2 enhancer.

Characterization of Cationic Bolaamphiphile Vesicles for siRNA Delivery into Tumors and Brain.

Small interfering RNAs (siRNAs) are potential therapeutic substances due to their gene silencing capability as exemplified by the recent approval by the US Food and Drug Administration (FDA) of the first siRNA therapeutic agent (patisiran). However, the delivery of naked siRNAs is challenging because of their short plasma half-lives and poor cell penetrability. In this study, we used vesicles made from bolaamphiphiles (bolas), GLH-19 and GLH-20, to investigate their ability to protect siRNA from degradation by nucleases while delivering it to target cells, including cells in the brain. Based on computational and experimental studies, we found that GLH-19 vesicles have better delivery characteristics than do GLH-20 vesicles in terms of stability, binding affinity, protection against nucleases, and transfection efficiency, while GLH-20 vesicles contribute to efficient release of the delivered siRNAs, which become available for silencing. Our studies with vesicles made from a mixture of the two bolas (GLH-19 and GLH-20) show that they were able to deliver siRNAs into cultured cancer cells, into a flank tumor and into the brain. The vesicles penetrate cell membranes and the blood-brain barrier (BBB) by endocytosis and transcytosis, respectively, mainly through the caveolae-dependent pathway. These results suggest that GLH-19 strengthens vesicle stability, provides protection against nucleases, and enhances transfection efficiency, while GLH-20 makes the siRNA available for gene silencing.

Author Correction: C/EBPß regulates delta-secretase expression and mediates pathogenesis in mouse models of Alzheimer's disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulation of senescence and the SASP by the transcription factor C/EBPß.

Oncogene-induced senescence (OIS) serves as an important barrier to tumor progression in cells that have acquired activating mutations in RAS and other oncogenes. Senescent cells also produce a secretome known as the senescence-associated secretory phenotype (SASP) that includes pro-inflammatory cytokines and chemokines. SASP factors reinforce and propagate the senescence program and identify senescent cells to the immune system for clearance. The OIS program is executed by several transcriptional effectors that include p53, RB, NF-κB and C/EBPß. In this review, we summarize the critical role of C/EBPß in regulating OIS and the SASP. Post-translational modifications induced by oncogenic RAS signaling control C/EBPß activity and dimerization, and these alterations switch C/EBPß to a pro-senescence form during OIS. In addition, C/EBPß is regulated by a unique 3'UTR-mediated mechanism that restrains its activity in tumor cells to facilitate senescence bypass and suppression of the SASP.

C/EBPß regulates delta-secretase expression and mediates pathogenesis in mouse models of Alzheimer's disease.

Delta-secretase cleaves both APP and Tau to mediate the formation of amyloid plaques and neurofibrillary tangle in Alzheimer's disease (AD). However, how aging contributes to an increase in delta-secretase expression and AD pathologies remains unclear. Here we show that a CCAAT-enhancer-binding protein (C/EBPß), an inflammation-regulated transcription factor, acts as a key age-dependent effector elevating both delta-secretase (AEP) and inflammatory cytokines expression in mediating pathogenesis in AD mouse models. We find that C/EBPß regulates delta-secretase transcription and protein levels in an age-dependent manner. Overexpression of C/EBPß in young 3xTg mice increases delta-secretase and accelerates the pathological features including cognitive dysfunctions, which is abolished by inactive AEP C189S. Conversely, depletion of C/EBPß from old 3xTg or 5XFAD mice diminishes delta-secretase and reduces AD pathologies, leading to amelioration of cognitive impairment in these AD mouse models. Thus, our findings support that C/EBPß plays a pivotal role in AD pathogenesis via increasing delta-secretase expression.

A RAS-CaMKKß-AMPKα2 pathway promotes senescence by licensing post-translational activation of C/EBPß through a novel 3'UTR mechanism.

Oncogene-induced senescence (OIS) is an intrinsic tumor suppression mechanism that requires the p53 and RB pathways and post-translational activation of C/EBPß through the RAS-ERK cascade. We previously reported that in transformed/proliferating cells, C/EBPß activation is inhibited by G/U-rich elements (GREs) in its 3'UTR. This mechanism, termed "3'UTR regulation of protein activity" (UPA), maintains C/EBPß in a low-activity state in tumor cells and thus facilitates senescence bypass. Here we show that C/EBPß UPA is overridden by AMPK signaling. AMPK activators decrease cytoplasmic levels of the GRE binding protein HuR, which is a key UPA component. Reduced cytoplasmic HuR disrupts 3'UTR-mediated trafficking of Cebpb transcripts to the peripheral cytoplasm-a fundamental feature of UPA-thereby stimulating C/EBPß activation and growth arrest. In primary cells, oncogenic RAS triggers a Ca++-CaMKKß-AMPKα2-HuR pathway, independent of AMPKα1, that is essential for C/EBPß activation and OIS. This axis is disrupted in cancer cells through down-regulation of AMPKα2 and CaMKKß. Thus, CaMKKß-AMPKα2 signaling constitutes a key tumor suppressor pathway that activates a novel UPA-cancelling mechanism to unmask the cytostatic and pro-senescence functions of C/EBPß.

IKKα inactivation promotes Kras-initiated lung adenocarcinoma development through disrupting major redox regulatory pathways.

Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two distinct and predominant types of human lung cancer. IκB kinase α (IKKα) has been shown to suppress lung SCC development, but its role in ADC is unknown. We found inactivating mutations and homologous or hemizygous deletions in the CHUK locus, which encodes IKKα, in human lung ADCs. The CHUK deletions significantly reduced the survival time of patients with lung ADCs harboring KRAS mutations. In mice, lung-specific Ikkα ablation (IkkαΔLu ) induces spontaneous ADCs and promotes KrasG12D-initiated ADC development, accompanied by increased cell proliferation, decreased cell senescence, and reactive oxygen species (ROS) accumulation. IKKα deletion up-regulates NOX2 and down-regulates NRF2, leading to ROS accumulation and blockade of cell senescence induction, which together accelerate ADC development. Pharmacologic inhibition of NADPH oxidase or ROS impairs KrasG12D-mediated ADC development in IkkαΔLu mice. Therefore, IKKα modulates lung ADC development by controlling redox regulatory pathways. This study demonstrates that IKKα functions as a suppressor of lung ADC in human and mice through a unique mechanism that regulates tumor cell-associated ROS metabolism.

RNA Fibers as Optimized Nanoscaffolds for siRNA Coordination and Reduced Immunological Recognition.

RNA is a versatile biomaterial that can be used to engineer nanoassemblies for personalized treatment of various diseases. Despite promising advancements, the design of RNA nanoassemblies with minimal recognition by the immune system remains a major challenge. Here, an approach is reported to engineer RNA fibrous structures to operate as a customizable platform for efficient coordination of siRNAs and for maintaining low immunostimulation. Functional RNA fibers are studied in silico and their formation is confirmed by various experimental techniques and visualized by atomic force microscopy (AFM). It is demonstrated that the RNA fibers offer multiple advantages among which are: i) programmability and modular design that allow for simultaneous controlled delivery of multiple siRNAs and fluorophores, ii) reduced immunostimulation when compared to other programmable RNA nanoassemblies, and iii) simple production protocol for endotoxin-free fibers with the option of their cotranscriptional assembly. Furthermore, it is shown that functional RNA fibers can be efficiently delivered with various organic and inorganic carriers while retaining their structural integrity in cells. Specific gene silencing triggered by RNA fibers is assessed in human breast cancer and melanoma cell lines, with the confirmed ability of functional fibers to selectively target single nucleotide mutations.

Oncogenic RAS-Induced Perinuclear Signaling Complexes Requiring KSR1 Regulate Signal Transmission to Downstream Targets.

The precise characteristics that distinguish normal and oncogenic RAS signaling remain obscure. Here, we show that oncogenic RAS and BRAF induce perinuclear relocalization of several RAS pathway proteins, including the kinases CK2 and p-ERK1/2 and the signaling scaffold KSR1. This spatial reorganization requires endocytosis, the kinase activities of MEK-ERK and CK2, and the presence of KSR1. CK2α colocalizes with KSR1 and Rab11, a marker of recycling endosomes, whereas p-ERK associates predominantly with a distinct KSR1-positive endosomal population. Notably, these perinuclear signaling complexes (PSC) are present in tumor cell lines, mouse lung tumors, and mouse embryonic fibroblasts undergoing RAS-induced senescence. PSCs are also transiently induced by growth factors (GF) in nontransformed cells with delayed kinetics (4-6 hours), establishing a novel late phase of GF signaling that appears to be constitutively activated in tumor cells. PSCs provide an essential platform for RAS-induced phosphorylation and activation of the prosenescence transcription factor C/EBPß in primary MEFs undergoing senescence. Conversely, in tumor cells, C/EBPß activation is suppressed by 3'UTR-mediated localization of Cebpb transcripts to a peripheral cytoplasmic domain distinct from the PSC region. Collectively, our findings indicate that sustained PSC formation is a critical feature of oncogenic RAS/BRAF signaling in cancer cells that controls signal transmission to downstream targets by regulating selective access of effector kinases to substrates such as C/EBPß.Significance: In addressing the long-standing question of the difference between normal and oncogenic RAS pathway signaling, this study shows that oncogenic RAS specifically triggers constitutive endocytosis-dependent movement of effector kinases to a perinuclear region, thereby creating connections to unique downstream targets such as the core prosenescence and the inflammatory regulatory transcription factor C/EBPß. Cancer Res; 78(4); 891-908. ©2017 AACR.

Dual negative roles of C/EBPα in the expansion and pro-tumor functions of MDSCs.

Myeloid-derived suppressor cells (MDSCs) are greatly expanded in cancer patients and tumor-bearing mice. They infiltrate into tumors and modulate the tumor microenvironment. In an effort to identify molecular mediators responsible for expansion and the tumor-promoting function of MDSCs, we discovered CCAAT/enhancer binding protein alpha (C/EBPα) expression was significantly reduced in MDSCs from tumor-bearing mice compared to non-tumor-bearing hosts. Tumor-conditioned medium down-regulated C/EBPα expression, suggesting tumor secreted factors inhibiting the gene expression. Consistent with the function of C/EBPα in regulating the balance between proliferation and growth arrest in hematopoietic progenitors, myeloid lineage specific deletion of C/EBPα resulted in significantly enhanced MDSC proliferation and expansion, as well as an increase of myeloid progenitors and a decrease of mature cells. In addition, deletion of C/EBPα in MDSCs enhanced the pro-angiogenic, immune suppressive and pro-tumorigenic behavior of these cells by upregulating the production of iNOS and arginase, as well as MMP-9 and VEGF. Accordingly, tumors growing in C/EBPα conditional null mice displayed greater MDSC infiltration, increased vascularization and accelerated tumor growth. Taken together, this study reveals dual negative roles of C/EBPα in the expansion as well as pro-angiogenic and immune suppressive functions in MDSCs.

Identification of senescent cell surface targetable protein DPP4.

Senescent cell accumulation in aging tissues is linked to age-associated diseases and declining function, prompting efforts to eliminate them. Mass spectrometry analysis revealed that DPP4 (dipeptidyl peptidase 4) was selectively expressed on the surface of senescent, but not proliferating, human diploid fibroblasts. Importantly, the differential presence of DPP4 allowed flow cytometry-mediated isolation of senescent cells using anti-DPP4 antibodies. Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) assays revealed that the cell surface DPP4 preferentially sensitized senescent, but not dividing, fibroblasts to cytotoxicity by natural killer cells. In sum, the selective expression of DPP4 on the surface of senescent cells enables their preferential elimination.

LPS independent activation of the pro-inflammatory receptor Trem1 by C/EBPε in granulocytes.

C/EBPε is a critical transcriptional factor for granulocyte differentiation and function. Individuals with germline mutations of C/EBPε fail to develop normal granulocytes and suffer from repeated infections. In order to gain a global view of the transcriptional machinery regulated by C/EBPε, we performed whole-genome ChIP-Seq using mouse bone marrow cells. To complement the C/EBPε DNA binding analyses, RNA-Sequencing was done in parallel using sorted mature and immature granulocytes from WT and C/EBPε KO bone marrow. This approach led to the identification of several direct targets of C/EBPε, which are potential effectors of its role in granulocytic differentiation and function. Interestingly, Trem1, a gene critical to granulocyte function, was identified as a direct C/EBPε target gene. Trem1 expression overlaps very closely with expression signature of C/EBPε during hematopoietic development. Luciferase reporter and EMSA assays revealed that C/EBPε binds to the regulatory elements of Trem1 and regulates its expression during granulocytic differentiation. In addition, we provide evidence that inflammatory stimuli (LPS) can also control the expression of Trem1 independent of C/EBPε. Overall, this study provides comprehensive profiling of the transcriptional network controlled by C/EBPε during granulopoiesis and identifies Trem1 as one of its downstream effectors involved in eliciting an immune response.

The Yersinia pestis Effector YopM Inhibits Pyrin Inflammasome Activation.

Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1ß and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1ß/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1ß/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1ß activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1ß generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1ß/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague.

C/EBPγ Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4.

The integrated stress response (ISR) controls cellular adaptations to nutrient deprivation, redox imbalances, and endoplasmic reticulum (ER) stress. ISR genes are upregulated in stressed cells, primarily by the bZIP transcription factor ATF4 through its recruitment to cis-regulatory C/EBP:ATF response elements (CAREs) together with a dimeric partner of uncertain identity. Here, we show that C/EBPγ:ATF4 heterodimers, but not C/EBPß:ATF4 dimers, are the predominant CARE-binding species in stressed cells. C/EBPγ and ATF4 associate with genomic CAREs in a mutually dependent manner and coregulate many ISR genes. In contrast, the C/EBP family members C/EBPß and C/EBP homologous protein (CHOP) were largely dispensable for induction of stress genes. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferate poorly and exhibit oxidative stress due to reduced glutathione levels and impaired expression of several glutathione biosynthesis pathway genes. Cebpg(-/-) mice (C57BL/6 background) display reduced body size and microphthalmia, similar to ATF4-null animals. In addition, C/EBPγ-deficient newborns die from atelectasis and respiratory failure, which can be mitigated by in utero exposure to the antioxidant, N-acetyl-cysteine. Cebpg(-/-) mice on a mixed strain background showed improved viability but, upon aging, developed significantly fewer malignant solid tumors than WT animals. Our findings identify C/EBPγ as a novel antioxidant regulator and an obligatory ATF4 partner that controls redox homeostasis in normal and cancerous cells.

An Arf-Egr-C/EBPß pathway linked to ras-induced senescence and cancer.

Oncogene-induced senescence (OIS) protects normal cells from transformation by Ras, whereas cells lacking p14/p19(Arf) or other tumor suppressors can be transformed. The transcription factor C/EBPß is required for OIS in primary fibroblasts but is downregulated by H-Ras(V12) in immortalized NIH 3T3 cells through a mechanism involving p19(Arf) loss. Here, we report that members of the serum-induced early growth response (Egr) protein family are also downregulated in 3T3(Ras) cells and directly and redundantly control Cebpb gene transcription. Egr1, Egr2, and Egr3 recognize three sites in the Cebpb promoter and associate transiently with this region after serum stimulation, coincident with Cebpb induction. Codepletion of all three Egrs prevented Cebpb expression, and serum induction of Egrs was significantly blunted in 3T3(Ras) cells. Egr2 and Egr3 levels were also reduced in Ras(V12)-expressing p19(Arf) null mouse embryonic fibroblasts (MEFs), and overall Egr DNA-binding activity was suppressed in Arf-deficient but not wild-type (WT) MEFs, leading to Cebpb downregulation. Analysis of human cancers revealed a strong correlation between EGR levels and CEBPB expression, regardless of whether CEBPB was increased or decreased in tumors. Moreover, overexpression of Egrs in tumor cell lines induced CEBPB and inhibited proliferation. Thus, our findings identify the Arf-Egr-C/EBPß axis as an important determinant of cellular responses (senescence or transformation) to oncogenic Ras signaling.

Structural insights into interactions of C/EBP transcriptional activators with the Taz2 domain of p300.

Members of the C/EBP family of transcription factors bind to the Taz2 domain of p300/CBP and mediate its phosphorylation through the recruitment of specific kinases. Short sequence motifs termed homology boxes A and B, which comprise their minimal transactivation domains (TADs), are conserved between C/EBP activators and are necessary for specific p300/CBP binding. A possible mode of interaction between C/EBP TADs and the p300 Taz2 domain was implied by the crystal structure of a chimeric protein composed of residues 1723-1818 of p300 Taz2 and residues 37-61 of C/EBPℇ. The segment corresponding to the C/EBPℇ TAD forms two orthogonally disposed helices connected by a short linker and interacts with the core structure of Taz2 from a symmetry-related molecule. It is proposed that other members of the C/EBP family interact with the Taz2 domain in the same manner. The position of the C/EBPℇ peptide on the Taz2 protein interaction surface suggests that the N-termini of C/EBP proteins are unbound in the C/EBP-p300 Taz2 complex. This observation is in agreement with the known location of the docking site of protein kinase HIPK2 in the C/EBPß N-terminus, which associates with the C/EBPß-p300 complex.

C/EBPγ suppresses senescence and inflammatory gene expression by heterodimerizing with C/EBPß.

C/EBPß is an important regulator of oncogene-induced senescence (OIS). Here, we show that C/EBPγ, a heterodimeric partner of C/EBPß whose biological functions are not well understood, inhibits cellular senescence. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferated poorly, entered senescence prematurely, and expressed a proinflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPß. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPß. Covalently linked C/EBPß homodimers (ß∼ß) inhibited the proliferation and tumorigenicity of Ras(V12)-transformed NIH 3T3 cells, activated SASP gene expression, and recruited the CBP coactivator in a Ras-dependent manner, whereas Î³âˆ¼ß heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg(-/-) MEFs. C/EBPß depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPß homodimers in Cebpg(-/-) MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg(-/-) bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPß through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes.