senescence from d12 on (fig. S18, C to F).
However, small clusters of Tom+KRASG12V
hepatocytes were observed in d28 livers with
much higher frequency whenp21was inac-
tivated (Fig. 6F). Hepatocytes within these
clusters were cycling at a markedly higher
rate than corresponding hepatocytes located
in isolation (Fig. 6G). Thus, p21-dependent
immunoclearance of cells that experience
oncogenic stress constitutes an important
first line of defense against neoplastic growth.
p21 engages macrophages to set a biological
timer that controls cell fate
Stress-inducing oncogenic point mutations
are irreparable, but many cellular stresses are
transient or repairable. To determine whether
stressed cells that recuperate and normalize
p21 cease to produce a PASP and are released
from immunosurveillance, we produced MEFs
containing a lentiviral construct that allows
for doxycycline (dox)–inducible expression of
p21-Myc-Flag. These MEFs stopped prolifer-
ating within 2 days after dox administration,
but were fully capable of resuming the cell
cycle when p21 levels normalized upon dox
withdrawal (Fig. 7, A and B, and fig. S19, A
to C). CM harvested from d2 p21-OE MEFs
stimulated fibroblast migration in the scratch
assay and macrophage migration in the trans-
well assay with peritoneal immune cells (Fig.
7Candfig.S19,DandE).Bycontrast,CM
prepared from MEFs that had been on dox
for 2 days followed by 4 days off dox had no
impact on cell migratory properties in these
same assays.Cxcl14expression followed the
promigratory properties of p21-OE CM, with
reducedCxcl14expression upon withdrawal
corresponding to a more generalized collapse
in PASP gene expression (Fig. 7D). Suppres-
sion of E2F target genes, proliferative arrest,
induction of PASP genes, and chemoattraction
of macrophages all occurred within 24 hours
after induction of p21 (fig. S19, F to J). Thus,
the brake on cell-cycle progression and PASP-
mediated immune surveillance occur simulta-
neously and rapidly.
To determine the time allocated to damaged
cells to recuperate and avert elimination by
immune cells under physiological conditions
and to define the underlying timer mecha-
nism, we created transgenic mice in which p21
could be temporally overexpressed in hepato-
cytes by sequential administration of adeno-Cre injection and dox (Fig. 8A). Macrophages
surrounding hepatocytes overexpressing p21
for up to 4 days promptly withdrew upon nor-
malization of p21 (Fig. 8, B and C, and fig. S20,
A and B). By contrast, when p21 levels were
normalized at day 6, macrophages surround-
ing hepatocytes failed to disengage. At this
stage, hepatocytes were adjoined not only by
macrophages that had undergone M1 acti-
vation, but also lymphocytes that had been
primed for target cell elimination (Fig. 8, D
to F). At d6, p21-OE hepatocytes were not yet
senescent, although some entry into the senes-
cent state occurred during the 2-day off period
(fig. S20, C to E). Thus, p21 induction sets a
timeframe for stress repair or adaptation that
is defined by the time it takes for the immune
system to transition from a cell-surveillance to
a cell-clearance mode.Discussion
In response to a wide variety of stress stimuli,
p53 induces p21 to halt the cell cycle as one of
the hundreds of downstream p53 transcrip-
tional targets. As such, p21 provides time for
cells to recuperate through various repair and
adaptation mechanisms, or if the damage is
too severe, for induction of apoptosis or for
cellular senescence. Our studies revealed that
p21 also responds to cellular stressors through
a cell-nonautonomous mechanism by placing
cells under immunosurveillance and that p21
does so concomitantly with halting cell-cycle
progression. These findings extend earlier evi-
dence linking p21 inactivation to immune sys-
tem deficiencies and predisposition to systemic
autoimmunity ( 29 – 31 ). In probing the mechan-
ism, we discovered that the pool of hypophos-
phorylated Rb that is created in response to
p21 induction binds to chromatin to not only
establish cell-cycle arrest but also to cooperate
with select SMAD and STAT TFs to create a
bioactive secretome with diverse biological
functions, including immunosurveillance.
We found that CXCL14 within this secretome
is instrumental in attracting macrophages to
cells with elevated p21. By recruiting macro-
phages, p21 sets a biological timer that allows
for a period of repair or adaptation of about
4 days (at least for hepatocytes), which expires
when macrophages polarize toward an M1
phenotype and lymphocytes are recruited to
mount a cytotoxic T cell response. Therefore,
the p21-mediated killing of stressed cells by
the leveraging of the immune system represents
an important complementary mechanism to
p53-mediated apoptosis. Hepatocytes that
induce p21 in response to a well-established
oncogenic stressor that drives hepatocellular
carcinoma are eliminated via this mechanism,
underscoring its physiological relevance. Thus,
the first line of tumor-protective immuno-
surveillance appears to already occur at the
earliest stages of cellular transformation,Sturmlechneret al.,Science 374 , eabb3420 (2021) 29 October 2021 6 of 15
Fig. 5. p21 induced
by oncogenic RAS places
cells under immunosur-
veillance.(A) (Top)
Schematic representation
ofL-KRASG12VandAi14
transgenes, andp21-and
Rb-conditional knockout
alleles. Blue triangles
denote LoxP sites.
(Bottom) Schematic of
the experimental design.
(B) Proportion of Tom+
p21+hepatocytes among
Tom+hepatocytes at
indicated days after
adeno-Cre injection.
(C) Quantification of Tom+
hepatocytes joined by
≥3 F4/80+macrophages.
p21hi, cells with elevated
p21 staining; p21lo,
cells with baseline or
background level p21
staining. Mice used were
on a C57BL/6×129Sv
mixed genetic background.
Data represent means ±
SEM. Experiments in
the above panels were
performed once. ns, not
significant. ***P< 0.001.
Two-way ANOVA with
Sidak’s correction
[d12 and d28 in (B)], one-way ANOVA with Sidak’s correction [d4 in (B) and (C)].
A- Ai14
- Ai14;L-KRASG12V
- Ai14;L-KRASG12V;p21f/f
- Ai14;L-KRASG12V;Rbf/f
Tom+hepatocytes
Tom+;KRASG12Vhepatocytes
Tom+;KRASG12V;p21-KO hepatocytes
Tom+;KRASG12V;Rb-KO hepatocytesAdeno-CreL-KRASG12VExon 2 p21floxedCdkn1alocusExon 19 RbfloxedRb1locusBd4 d12 d28Tom+
p21+
hepatocytes (%)****** ***ns ns
nsnsns************ns***
***
nsnsd4 Tom+
hepatocytes joinedby3 F4/80+
cells (%)Ai14 (n=5)
Ai14;L-KRASG12V(n=5)
Ai14;L-KRASG12V;p21f/f (n=5)
Ai14;L-KRASG12V;Rbf/f (n=5)C******pCAG STOP WPREpCAG STOP tdTomato WPREAi14KRASG12V-Mycp21lo
p21lo
p21hi
p21lo
p21hi
p21(^0) lo
10
20
30
50
40
0
25
50
75
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