RESEARCH ARTICLE
◥IMMUNOLOGY
p21 produces a bioactive secretome that places
stressed cells under immunosurveillance
Ines Sturmlechner1,2, Cheng Zhang^3 , Chance C. Sine^1 , Erik-Jan van Deursen^1 , Karthik B. Jeganathan^1 ,
Naomi Hamada^1 , Jan Grasic^1 , David Friedman^4 , Jeremy T. Stutchman^1 , Ismail Can^5 ,
Masakazu Hamada^1 , Do Young Lim^1 , Jeong-Heon Lee^6 , Tamas Ordog6,7, Remi-Martin Laberge^8 ,
Virginia Shapiro^4 , Darren J. Baker1,5, Hu Li^3 , Jan M. van Deursen1,5
Immune cells identify and destroy damaged cells to prevent them from causing cancer or other pathologies
by mechanisms that remain poorly understood. Here, we report that the cell-cycle inhibitor p21 places cells
under immunosurveillance to establish a biological timer mechanism that controls cell fate. p21 activates
retinoblastoma protein (Rb)Ðdependent transcription at select gene promoters to generate a complex
bioactive secretome, termed p21-activated secretory phenotype (PASP). The PASP includes the chemokine
CXCL14, which promptly attracts macrophages. These macrophages disengage if cells normalize p21
within 4 days, but if p21 induction persists, they polarize toward an M1 phenotype and lymphocytes mount
a cytotoxic T cell response to eliminate target cells, including preneoplastic cells. Thus, p21 concurrently
induces proliferative arrest and immunosurveillance of cells under duress.
C
ells in complex multicellular organisms
are subject to a myriad of stresses,
which can be managed through vari-
ous cell-autonomous adaptation and
repair mechanisms ( 1 ). Cells that fail
to recuperate—for instance, because of severe
or prolonged stresses—engage programs that
execute regulated cell death or cellular senes-
cence, thereby limiting the risk of neoplastic
transformation ( 2 , 3 ). The cellular senescence
program is characterized by permanent cell-
cycle withdrawal through the induction of
cyclin-dependent kinase inhibitors like p21
and p16, encoded byp21(CDKN1A) andp16
(CDKN2A), respectively ( 4 – 7 ). In mammals,
cellular senescence has also been implicated in
biological processes beyond cancer, including
development( 8 , 9 ), tissue repair ( 10 ), aging,
and age-related disorders ( 3 , 11 – 15 ). Senes-
cent cells presumably exert these functions
by virtue of the senescence-associated secre-
tory phenotype (SASP), a diverse collection
of secreted factors (SFs) often enriched in
immune-modulatory cytokines and chemo-
kines, matrix remodeling enzymes, and growth
factors ( 5 , 16 – 18 ). Once established, senescent
cells can persist in tissues and organs for pro-
longed periods of time ( 19 ), although they can
also be recognized and eliminated by lym-
phocytes through mechanisms that remain
ill-defined ( 4 , 16 , 20 ).
We sought to better understand the proper-
ties of senescent cells at a molecular mecha-
nistic level by identifying senescence-associated
super-enhancer (SASE)–controlled genes. Super-
enhancers are large enhancers that are highly
enriched in certain chromatin modifications
and bind large amounts of numerous tran-
scription factors (TFs) and coactivators ( 21 , 22 ).
Super-enhancers regulate genes with impor-
tant functions in processes that are cell type–
specific or define cell identity ( 23 , 24 ), which
led us to hypothesize that the identification
and in-depth characterization of genes that
come under the control of super-enhancers
with senescence would be particularly inform-
ative about the inner workings of this cell fate.
In pursuing this idea, we focused our efforts
on genes controlled by SASEs that are high-
ly conserved across species, cell types, and
senescence-inducing stressors. Our study illu-
minates that immediate-early cell-autonomous
reactions to cellular stress are coordinated with
cell nonautonomous responses through p21-
mediated changes in the Rb-dependent tran-
scriptional landscape to establish a biological
timer mechanism for cell fate decisions.p21 regulates the SASP through
Rb-dependent transcription
In our initial screen, we exposed primary mouse
embryonic fibroblasts (MEFs) to three distinct
senescence-inducing stressors:g-irradiation
(IR), extensive replication (REP), and oncogene-induced (OI) signaling by overexpression of
human KRASG12V(fig. S1). We mapped the
common super-enhancer changes as these
cells transitioned to a senescent state, and
identified the transcriptionally activated genes
associated with these super-enhancers (fig. S2,
A and B). We uncovered 50 such genes (fig.
S2B and tables S1 and S2), three of which were
also associated with a SASE in IR-senescent
human fetal lung (IMR-90) cells and tran-
scriptionally up-regulated in both IR- and OI-
senescent IMR-90 cells, includingp21(fig. S2,
A to C, and table S3). Notably, H3K27Ac chro-
matin immunoprecipitation–quantitative
polymerase chain reaction (ChIP-qPCR) on
OI-senescent cells (SNCs) collected from mouse
liver indicated that the SASE identified near the
p21locus was conserved in vivo (fig. S2, D to J).
Fully SNCs deficient in p21 incorporated
5-ethynyl-2′-deoxyuridine (EdU) (fig. S3, A to
D), indicating that sustaining p21 in the senes-
cent state is important to prevent cell-cycle
reentry through continued transcriptional
repression of E2F target genes via hypophos-
phorylation of Rb ( 25 ). Intriguingly, knocking
downp21in SNCs also decreased expression
of multiple SASP factors as determined by
reverse transcription (RT)–qPCR for a panel
of well-established SASP factors (fig. S3E).
Comprehensive transcriptomic analysis of
IR-senescent MEFs using RNA sequencing
(RNA-seq) revealed that about a third of the
SASP (188 of 503 factors) was p21-dependent
(Fig. 1A; fig. S4, A to C; and table S4). Similarly,
nearly half the SASP (167 of 354 factors) iden-
tified in IR-senescent IMR-90 cells was depen-
dentonp21(Fig.1A,fig.S4D,andtableS4),
which prompted us to probe the mechanism(s)
underlying these p21-dependent secretory
phenotypes, hereafter referred to as the p21-
activated secretory phenotype (PASP).
We first focused on Rb and found that its
absence in SNCs not only activated E2F target
genes (figs. S3, F to I, and S5) but also de-
creased expression of most of the SASP factors
down-regulated withp21deficiency (Fig. 1, A
and B; fig. S3J; and table S4), suggesting that
p21 confers its effect on the SASP through
hypophosphorylation of Rb. To explore how
p21-mediated Rb hypophosphorylation might
activate SASP genes, we identified TFs that
have been linked to the SASP, inflammation, or
cytokine production and used their transcrip-
tional targets in overrepresentation analyses
on RNA-seq data from IR-, REP, OI-senescent
MEFs, IR-senescent IMR-90 cells, and their non-
senescent counterparts. We found that RELA,
CEBPb, SMAD2, SMAD3, STAT1, STAT5A/B,
and STAT6 were consistently more transcrip-
tionally active in SNCs than in non-SNCs re-
gardless of senescence-inducing stressor or
species(Fig.1C).RELA,SMAD2,SMAD3,STAT1,
and STAT6 lost this status whenp21orRbwas
depleted (Fig. 1C). Thus, hypophosphorylatedRESEARCH
Sturmlechneret al.,Science 374 , eabb3420 (2021) 29 October 2021 1of15
(^1) Department of Pediatric and Adolescent Medicine, Mayo
Clinic, Rochester, MN, USA.^2 Department of Pediatrics,
Molecular Genetics Section, University of Groningen,
University Medical Center Groningen, Antonius Deusinglaan
1, 9713 AV Groningen, Netherlands.^3 Department of
Molecular Pharmacology and Experimental Therapeutics,
Mayo Clinic, Rochester, MN, USA.^4 Department of
Immunology, Mayo Clinic, Rochester, MN, USA.^5 Department
of Biochemistry and Molecular Biology, Mayo Clinic,
Rochester, MN, USA.^6 Epigenomics Program, Center for
Individualized Medicine, Mayo Clinic, Rochester, MN, USA.
(^7) Department of Physiology and Biomedical Engineering,
Mayo Clinic, Rochester, MN, USA.^8 Unity Biotechnology,
South San Francisco, CA 94080, USA.
*Corresponding author. Email: [email protected]
(J.M.v.D.); [email protected] (H.L.)