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ARTICLE
Lysine metabolism is a novel metabolic tumor suppressor
pathway in breast cancer
1✉
Jianchun Wu1, Kaitrin Kramer1 and David L. Crowe

© The Author(s), under exclusive licence to Springer Nature Limited 2023

The International Agency for Research on Cancer determined that obesity is the primary preventable cause of breast cancer. The nuclear
receptor peroxisome proliferator activated receptor γ (PPARγ) binds inflammatory mediators in obesity and its expression is reduced in
human breast cancer. We created a new model to better understand how the obese microenvironment alters nuclear receptor function
in breast cancer. The obesity related cancer phenotype was PPARγ dependent; deletion of PPARγ in mammary epithelium which is a
tumor suppressor in lean mice unexpectedly increased tumor latency, reduced the luminal progenitor (LP) tumor cell fraction, and
increased autophagic and senescent cells. Loss of PPARγ expression in mammary epithelium of obese mice increased expression of
2-aminoadipate semialdehyde synthase (AASS) which regulates lysine catabolism to acetoacetate. PPARγ-associated co-repressors and
activators regulated AASS expression via a canonical response element. AASS expression was significantly reduced in human breast
cancer, and AASS overexpression or acetoacetate treatment inhibited proliferation and induced autophagy and senescence in human
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breast cancer cell lines. Genetic or pharmacologic HDAC inhibition promoted autophagy and senescence in mammary tumor cells in
vitro and in vivo. We concluded that lysine metabolism is a novel metabolic tumor suppressor pathway in breast cancer.

Oncogene (2023) 42:2402–2414; https://doi.org/10.1038/s41388-023-02766-8

INTRODUCTION with increased incidence of clinically aggressive basal subtype

Over 280,000 cases of breast cancer are diagnosed in the
United States annually, and over 40,000 women will die of the
disease [1]. Important tumor suppressor genes such as BRCA1
have been identified in hereditary breast cancer [2], but these
tumors comprise a small group of clinical cases. Research in
breast cancer genetics has classified these tumors into specific
categories [3] which have implications for patient treatment.
Peroxisome proliferator activated receptors (PPAR) have func-
tional domains for ligand binding and interaction with response
elements in regulatory regions of their target genes to modulate
transcription [4]. This regulation is mediated by epigenetic histone
deacetylase (HDAC) repressors and histone acetyltransferase (HAT)
coactivators which modify chromatin structure of target genes.
Specific dietary lipids such as long chain saturated and unsaturated
fatty acids selectively activate PPARs [5]. Other inflammatory
mediators such leukotrienes and prostaglandins also are PPAR
ligands [6]. PPARγ expression is reduced in human breast cancer
consistent with its recognized tumor suppressor function [7, 8]. The
International Agency for Research on Cancer determined that
obesity is the primary preventable cause of breast cancer [9].
Obesity causes local and systemic inflammation that contribute to
the development and progression of breast cancer [10]. Obese
women have increased levels of pro-inflammatory mediators
which result in elevated risk of breast cancer [5, 11]. A positive
association between obesity and postmenopausal breast cancer
risk has been consistently observed in epidemiological studies
[12, 13]. Breast cancer prognosis is substantially worse for obese
compared to normal weight women [14, 15]. Obesity is associated
breast cancer [16].
We created a new genetic model in order to better understand
how the obese microenvironment alters nuclear receptor function in
breast cancer. Genetic deletion of PPARγ in mammary epithelium
which is a tumor suppressor in lean mice [8] surprisingly resulted in
long tumor latency and depleted LP tumor cells in the obese
microenvironment. The LP population is the cell of origin in basal
subtype breast cancer in humans [17, 18]. PPARγ inhibited expression
of the enzyme 2-aminoadipate semialdehyde synthase (AASS) which
catalyzes the rate limiting step in catabolism of the amino acid lysine
to the ketone body acetoacetate [19]. Our data demonstrated that
AASS expression is significantly reduced in human breast cancer.
Loss of PPARγ expression in the obese microenvironment resulted in
increased AASS expression and mammary tumor cell autophagy.
Autophagy is a catabolic process in which cells degrade their
proteins and organelles [20–22]. AASS overexpression or acetoace-
tate treatment induced autophagy and senescence in human breast
cancer cell lines. Inhibition of the PPAR co-repressor HDAC3 induced
autophagy and senescence in mammary tumors. Our results indicate
that lysine metabolism is a novel metabolic tumor suppressor
pathway in breast cancer.
RESULTS
Effects of PPARγ deficiency on mammary tumorigenesis in the
obese microenvironment
We examined the effects of PPARγ on mammary tumor
phenotype in obese genetically engineered mouse models.

University of Illinois Cancer Center, 801 S. Paulina Street, Room 525, Chicago, IL 60612, USA. ✉email: dlcrowe@uic.edu
1

Received: 5 February 2023 Accepted: 26 June 2023

Published online: 1 July 2023

MMTV-Cre;ppargf/f;mc4r−/−;Wnt obese mice lack PPARγ
expression in mammary epithelium. Wnt1 expression induces
basal subtype mammary tumors whose incidence is increased
in obese women. Melanocortin 4 receptor null mutant mice
(mc4r−/−) are hyperphagic and rapidly become obese on
standard diet (up to 70 g body weight compared to 20–25 g in
age matched littermate control animals). We predicted that
deleting PPARγ expression in obese mice would decrease
mammary tumor latency; however tumor latency in MMTV-
Cre;ppargf/f;mc4r−/−;Wnt mice increased to 168 days com-
pared to 105 days in MMTV-Cre;pparg+/+;mc4r−/−;Wnt
animals (p < 0.01; Fig. 1A). PPARγ expression in each genotype
is shown in Fig. 1B. Mammary tumors from MMTV-Cre;ppargf/
f;mc4r−/−;Wnt and MMTV-Cre;pparg+/+;mc4r−/−;Wnt mice
were histopathologically classified as poorly differentiated
adenocarcinomas (Fig. 1C, D). The tumorigenic LP cell popula-
tion was depleted in MMTV-Cre;ppargf/f;mc4r−/−;Wnt tumors
(0.6% vs. 3%; p < 0.008; Fig. 1E–G). The proliferating cell fraction
also was reduced in MMTV-Cre;ppargf/f;mc4r−/−;Wnt tumors
(12% vs. 37%; p < 0.03; Fig. 1H–J). The apoptotic cell fraction
was increased in MMTV-Cre;ppargf/f;mc4r−/−;Wnt tumors (9%
vs. 1.1%; p < 0.05; Fig. 1K–M). These results indicate that MMTV-
Cre;ppargf/f;mc4r−/−;Wnt mammary tumors exhibit increased
latency, reduced LP cell fraction, decreased proliferation, and
increased apoptotic cells compared to MMTV-Cre;
pparg+/+;mc4r−/−;Wnt cancers.
MMTV-Cre;ppargf/f;mc4r−/−;Wnt mammary tumors also
exhibited multiple autophagy and senescence markers. Trans-
mission electron microscopy demonstrated that MMTV-Cre;p-
pargf/f;mc4r−/−;Wnt tumor cells exhibited abundant
cytoplasmic vesicles consistent with autolysosome formation
(Fig. 2A, B). Expression of the autophagy marker LC3 [23]
increased in MMTV-Cre;ppargf/f;mc4r−/−;Wnt tumors (42% vs.
5%; p < 0.001; Fig. 2C, D, M). Western blot demonstrated that
MMTV-Cre;ppargf/f;mc4r−/−;Wnt mammary tumors expressed
increased PE-conjugated LC3 II relative to unconjugated LC3 I, a
specific marker of increased autophagy (3.2 vs. 5.8 ratio; Fig. 2E,
F; p < 0.04). We also examined co-localization of Lamp1+
lysosomes with Tom20+ mitochondria as an additional marker
of autolysosome formation in these tumors. The Lamp1+-
Tom20+ cell fraction increased in MMTV-Cre;ppargf/f;mc4r
−/−;Wnt mammary tumors (44% vs. 6%; p < 0.00002; Fig. 2G,
H, M). The cell fraction expressing the senescence marker
p16INK4A was increased in MMTV-Cre;ppargf/f;mc4r−/−;Wnt
mammary tumors (36% vs. 2%; p < 0.007; Fig. 2I, J, M). Similarly
the cell fraction exhibiting senescence associated heterochro-
matin foci (SAHF) was increased in MMTV-Cre;ppargf/f;mc4r
−/−;Wnt mammary tumors (49% vs. 10%; p < 0.0005; Fig.
2K–M). These results (increased tumor latency, progenitor cell
depletion, autophagy, and senescence) are consistent with
tumor suppressive cytostatic autophagy [24].
Increased lysine catabolism in PPARγ deficient mammary
tumors in the obese microenvironment
MMTV-Cre;ppargf/f;mc4r−/−;Wnt mammary tumors exhibited
16 fold increased expression of 2-aminoadipate semialdehyde
synthase mRNA (gene ID 30956; gene symbol Aass), the enzyme
regulating the rate limiting step in catabolism of lysine to
acetoacetate (p < 10−6; Fig. 3A). Increased expression of Aass
protein also was observed in mammary tumors from MMTV-
Cre;ppargf/f;mc4r−/−;Wnt (p < 0.00001; Fig. 3B, C), which was
associated with increased tumor acetoacetate levels (p < 0.002;
Figs. 3E and S1A, B). The acetoacetate mass spectrometry
standard is shown in Fig. S1C. Aass protein expression in
positive control tissue (liver) is shown in Fig. 3D. These results
indicate that MMTV-Cre;ppargf/f;mc4r−/−;Wnt mammary
tumors are characterized by increased Aass expression and
acetoacetate levels.
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PPARγ-mediated epigenetic regulation of the AASS gene in
mammary tumor cells
The AASS gene is located at mouse chromosome 6A3.1, and in
silico analysis demonstrated a canonical PPARγ response element
(PPRE; 5′- AGGTCAGAGGACA -3′) in intron 11 (chr6:23106215).
Chromatin immunoprecipitation demonstrated that PPARγ bound
to this site in mammary tumor derived LP cells but not in PPARγ
deficient LP cells (Fig. 4A). NCOR1, SMRT, and HDAC3 transcrip-
tional co-repressor proteins exhibited 4 fold decreased PPRE
association in LP cells from MMTV-Cre;ppargf/f;mc4r−/−;Wnt
tumors (p < 0.02). Acetylated histone H3K27, a marker of
transcriptional activation, exhibited 8 fold increased PPRE
association in LP cells from MMTV-Cre;ppargf/f;mc4r−/−;Wnt
(p < 0.007). We cloned 1.2 kb of intron 11 containing wild type
or mutated PPARγ binding site into the pGL3 luciferase reporter
vector which was transiently transfected into the mouse
mammary tumor cell line NF638. The PPRE sequence inhibited
reporter activity by 65%, and co-transfection with PPARγ, NCOR1,
SMRT, and HDAC3 expression vector further inhibited luciferase
activity by 72–95% (p < 0.0003; Fig. 4B). Conversely, CBP or PCAF
overexpression (p < 0.01) or treatment with the synthetic PPARγ
ligand rosiglitazone (p < 0.002) induced intron activity by 2 fold.
The mutated PPARγ binding site failed to repress luciferase activity
and was not affected by PPARγ expression. These results indicate
that PPARγ associated co-repressors and activators regulate AASS
expression via a canonical PPRE.
AASS expression in human breast cancers
In Gene Expression Omnibus (GEO) datasets (GDS3853), AASS
expression was significantly reduced in breast cancers and ductal
carcinomas in situ (DCIS) compared to normal breast epithelium
(p < 0.00008; Fig. 5A). Similar results were observed in another
GEO dataset (GDS2250; p < 0.0001; Fig. 5B), and in 1063 TCGA
breast cancer cases comprising luminal A, luminal B, basal, and
Her2 molecular classifications compared to 459 normal samples
(Fig. 5C; p < 0.04). We also examined AASS protein expression in
223 cases of breast cancer and histopathologically normal breast
samples by tissue microarrays. AASS protein expression was
detected in all histopathologically normal breast epithelium, but
was absent in 93% of breast cancer cases (p < 0.005; Fig. 5D–I).
AASS protein was undetectable in six human breast cancer cell
lines by western blot (Fig. 5J). These results indicate that AASS
expression is significantly reduced in human breast cancer.
Effects of AASS overexpression on human breast cancer cell
lines
To determine the effects of increased AASS expression, we
overexpressed this enzyme by stable transfection in human
breast cancer cell lines. AASS expression in MCF7 and MDA-MB-
468 experimental and control clones is shown in Fig. S2A.
Relative acetoacetate levels in AASS overexpressing breast
cancer clones were increased by 5 fold (p < 0.006; Fig. S2B).
Proliferation of AASS expressing clones decreased by 85%
compared to control (p < 0.001; Fig. S2C). AASS expressing
clones developed prominent cytoplasmic vacuoles which
gradually increased in size during the time course experiment
consistent with morphologic features of autophagy (Fig. S2D, E).
Expression of the autophagy marker LC3 II was increased 4–16
fold in AASS expressing clones (p < 10−8; Fig. S2A). A striking
increase in autophagic cells was observed in AASS expressing
human breast cancer cell lines as determined by Lamp1+-
Tom20+ autolysosome formation (75% vs. 1.4% in vehicle
treated cells; p < 10−7; Fig. S2F–H). The senescent cell fraction in
AASS expressing human breast cancer cells increased to 72%
(vs. 0.1% in control clones; p < 0.0002; Fig. S2I–K) as shown by
SAβgal staining. These results indicate that increased AASS
expression inhibits proliferation and induces autophagy and
senescence in human breast cancer cell lines.
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Effects of increased acetoacetate concentration on human
breast cancer cell lines
To determine the effects of increased acetoacetate on human
breast cancer cells, we treated cultured cell lines with acetoacetate
or isotonic saline vehicle solutions. At 1 mM acetoacetate
concentration, breast cancer cell lines developed prominent
cytoplasmic vacuoles which gradually increased in size during
the time course experiment consistent with morphologic features
of autophagy (Fig. S3A, B). This acetoacetate concentration is
within the physiologic range detected in human plasma samples.
A striking increase in Lamp1+Tom20+ autolysosomes was
observed in acetoacetate treated human breast cancer cell lines
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Fig. 1 Increased mammary tumor latency, LP cell depletion, decreased proliferation, and increased apoptosis in MMTV-Cre;ppargf/f;mc4r
−/−;Wnt mice. A Mammary tumor latency in MMTV-Cre;pparg+/+;mc4r−/−;Wnt and MMTV-Cre;ppargf/f;mc4r−/−;Wnt mice. Time in weeks is
shown on the x axis and percent tumor free mice is shown on the y axis. B PPARγ mRNA expression is shown in tumors from each genotype by qRT-
PCR. Histopathologic appearance of MMTV-Cre;pparg+/+;mc4r−/−;Wnt (C) and MMTV-Cre;ppargf/f;mc4r−/−;Wnt (D) mammary tumors by H&E
staining. Scale bar = 10 μm. LP cell fraction in MMTV-Cre;pparg+/+;mc4r−/−;Wnt (E) and MMTV-Cre;ppargf/f;mc4r−/−;Wnt (F) mammary tumors is
shown by flow cytometry. CD61 log scale and forward scatter linear scale is shown. G Quantitation of LP cell fraction in MMTV-Cre;pparg+/+;
mc4r−/−;Wnt and MMTV-Cre;ppargf/f;mc4r−/−;Wnt mammary tumors. Proliferating cell fraction in MMTV-Cre;pparg+/+;mc4r−/−;Wnt (H) and
MMTV-Cre;ppargf/f;mc4r−/−;Wnt (I) mammary tumors is shown by PCNA immunohistochemistry. Nuclei were counterstained with hematoxylin.
J Quantitation of PCNA+ cells in MMTV-Cre;pparg+/+;mc4r−/−;Wnt and MMTV-Cre;ppargf/f;mc4r−/−;Wnt mammary tumors. Error bars indicate
SEM. Apoptotic cell fraction in MMTV-Cre;pparg+/+;mc4r−/−;Wnt (K) and MMTV-Cre;ppargf/f;mc4r−/−;Wnt (L) mammary tumors is shown by
TUNEL analysis. Nuclei were counterstained with DAPI. M Quantitation of TUNEL+ cells in MMTV-Cre;pparg+/+;mc4r−/−;Wnt and MMTV-
Cre;ppargf/f;mc4r−/−;Wnt mammary tumors.

(85% vs. 0.1% in vehicle treated cells; p < 0.005; Fig. S3C–E). The
senescent cell fraction in acetoacetate treated human breast
cancer cells increased to 68% (vs. 0.2% in vehicle treated cells;
p < 0.002; Fig. S3F–H). Acetoacetate concentrations as low as 1 μM
inhibited proliferation of breast cancer cell lines, and this effect
was dose dependent (Fig. S3I). LC3 expression varied between
human breast cancer cell lines, but 2–4 fold increased expression
of phosphatidylethanolamine (PE)-conjugated LC3 II relative to
LC3 I was observed in acetoacetate treated MCF7, MDA-MB-231,
and MDA-MB-468 lines, indicating increased autophagy (p < 0.001;
Figs. S4 and S5). The LC3II/I ratio was 0.76 in vehicle treated MDA-
MB-468 cells which increased to 3.1 in the acetoacetate treated
group. Akt activation, which inhibits autophagy, was reduced by
4–18 fold in MCF7, T47D, and MDA-MB-231 cells (p < 0.0001).
pAMPK, an activator of autophagy, was increased by 3–4 fold in
MDA-MB-231 and MDA-MB-468 cell lines (p < 0.003). Expression of
Atg3, -5, -6, -7, -12, and -16 proteins varied between breast cancer
cell lines but was not affected by acetoacetate treatment. These
results indicate that increased acetoacetate concentrations inhibit
proliferation and induce autophagy and senescence in human
breast cancer cell lines.
Effects of HDAC inhibition on mammary tumorigenesis in vitro
and in vivo
Given that HDAC3 inhibited Aass gene activation, we hypothesized
that HDAC3 inhibition may upregulate AASS expression, increase
acetoacetate levels, and induce autophagy in mammary tumor
cells. We first inhibited HDAC3 expression by lentiviral shRNAs
transduction of LP cells from biologically aggressive mc4r−/−;Wnt
mammary tumors using in vitro tumorsphere cultures. HDAC3
expression was inhibited by 87% using shRNA transduction
(p < 0.0001; Fig. 6A). AASS expression was induced 18 fold in
mc4r−/−;Wnt tumorspheres transduced with HDAC3 shRNAs
(p < 0.0003; Fig. 6A). HDAC3 shRNAs transduction inhibited 7 day
tumorsphere growth in vitro (mean 65 vs. 147 μm diameter;
p < 0.01; Fig. 6B, C). HDAC3 shRNAs increased acetoacetate levels in
mc4r−/−;Wnt tumorspheres by 7 fold (p < 0.00008; Fig. 6D).
HDAC3 shRNAs increased expression of the autophagy marker
LC3 II in mc4r−/−;Wnt tumorspheres by 4 fold as shown by western
blot (p < 0.05; Fig. 6E). HDAC3 shRNAs increased the autophagic cell
fraction in mc4r−/−;Wnt tumorspheres (77% vs. 0.1%; p < 0.00003;
Fig. 6F–H) as shown by Lamp1/Tom20 immunofluorescence
microscopy. These results indicate that inhibiting HDAC3 expression
induces AASS expression, increases acetoacetate levels, inhibits
proliferation, and induces autophagy in mammary tumor cells.
To determine if HDAC inhibition was therapeutically effective
against mammary cancer in vivo, we next treated tumor bearing
mc4r−/−;Wnt mice with vorinostat. Vorinostat treated tumors
exhibited distinct hypocellularity by histopathology compared to
vehicle treated cancers (Fig. 7A, B). Vorinostat treatment reduced
proliferation of mammary tumor cells compared to vehicle treated
tumors (10 vs. 44%; p < 0.003; Fig. 7C–E). Vorinostat treatment
reduced the LP cell fraction (0.3 vs. 3.4%; p < 0.02; Fig. 7F, G).
Vorinostat treatment did not significantly increase the apoptotic
cell fraction compared to vehicle treated tumors (0.2%; Fig. 7H–J).
Vorinostat treatment increased relative acetoacetate levels by 3.2
fold compared to vehicle treated tumors (p < 0.01; Fig. 8A).
Vorinostat treatment increased LC3 expression compared to
vehicle treated tumors (74 vs. 0.1%; p < 0.0004; Fig. 8B, C, J).
Vorinostat treatment increased the autophagic cell fraction (80 vs.
0.2%; p < 10−7; Fig. 8D, E, J) compared to vehicle treated tumors.
Vorinostat treatment induced the expression of the senescence
marker p16INK4A compared to vehicle treated tumors (35 vs. 1%;
p < 0.0009; Fig. 8F, G, J). Vorinostat treatment induced SAHF
compared to vehicle treated tumors (64 vs. 0.1%; p < 10−6; Fig. 8H,
I, J). These results indicate that vorinostat treatment is effective in
inducing autophagy and senescence of mammary tumors.
DISCUSSION
Our previous study demonstrated that PPARγ deficiency induced
aggressive mammary tumors in lean mice consistent with its
tumor suppressor function [8]. Our current results demonstrated
that the PPARγ deficiency in the obese microenvironment resulted
in autophagy and senescence of mammary tumor cells. Previous
studies showed that PPARγ overexpression in cancer associated
fibroblasts (CAF) resulted in increased autophagy and senescence
[25, 26]. CAF induced senescence supported the growth of human
breast cancer cell lines [27]. Our results also demonstrated that
loss of PPARγ expression in the obese microenvironment inhibited
proliferation of mammary tumor cells. Similarly, cell cycle
inhibition in breast cancer and mammary epithelial cells also
increased autophagy [28]. These studies indicate that PPARγ is an
important regulator of cell proliferation, autophagy, and
senescence.
Our study characterized epigenetic regulation of a PPRE by
corepressors NCOR1, SMRT, and HDAC3 which regulated
expression the AASS gene in breast cancer cells (Fig. S6).
NCOR1 mutations were identified as genetic drivers in human
breast cancer and were associated with inflammatory and
metastatic tumors [29, 30]. SMRT was shown to repress CXCR4
expression via a PPRE in the gene promoter in human breast
cancer cell lines [31]. HDAC3 expression was correlated with
estrogen- and progesterone receptor negative breast cancers,
and with Her2 overexpression and poor survival [32]. Our study
also demonstrated that epigenetic coactivators such as CBP
induced AASS gene activity. CBP expression was increased in
human breast cancers compared to normal epithelium, and was
amplified in triple negative cases [33]. These studies indicate
that PPARγ-associated epigenetic corepressors and coactivators
are important regulators of breast cancer phenotype and
clinical outcomes.
Our results demonstrated that lysine catabolism to the ketone
body acetoacetate induced autophagy in human breast cancer
cell lines. We propose that increased AASS expression resulting in
elevated acetoacetate concentration in mammary tumors sug-
gests that there is no compensatory mechanism for converting
this excess acetoacetate to acetyl-CoA. Previous studies have

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Fig. 2 MMTV-Cre;ppargf/f;mc4r−/−;Wnt mammary tumors exhibit cytostatic autophagy and senescence. Autolysosome formation
(arrowheads) in MMTV-Cre;pparg+/+;mc4r−/−;Wnt (A) and MMTV-Cre;ppargf/f;mc4r−/−;Wnt (B) mammary tumors is shown by transmission
electron microscopy. Scale bar = 0.2 μm. Expression of the autophagy marker LC3 in MMTV-Cre;pparg+/+;mc4r−/−;Wnt (C) and MMTV-Cre;ppargf/
f;mc4r−/−;Wnt (D) mammary tumors is shown by immunohistochemistry. Nuclei were counterstained with hematoxylin. Scale bar = 10 μm.
E Increased expression of PE-conjugated LC3 II relative to LC3 I in MMTV-Cre;pparg+/+;mc4r−/−;Wnt and MMTV-Cre;ppargf/f;mc4r−/−;Wnt
mammary tumors is shown by western blot. β-actin expression was used as the protein loading control. F Quantitation of LC3 I and II expression in
MMTV-Cre;pparg+/+;mc4r−/−;Wnt and MMTV-Cre;ppargf/f;mc4r−/−;Wnt mammary tumors. Cytoplasmic co-localization of Lamp1+ lysosomes
with Tom20+ mitochondria in MMTV-Cre;pparg+/+;mc4r−/−;Wnt (G) and MMTV-Cre;ppargf/f;mc4r−/−;Wnt (H) mammary tumors from obese mice
is shown by immunofluorescence microscopy. Nuclei were counterstained with DAPI. Scale bar = 20 μm. Expression of the senescence marker
p16INK4A in MMTV-Cre;pparg+/+;mc4r−/−;Wnt (I) and MMTV-Cre;ppargf/f;mc4r−/−;Wnt (J) mammary tumors is shown by immunohistochemistry.
Senescence associated heterochromatin foci (SAHF) in MMTV-Cre;pparg+/+;mc4r−/−;Wnt (K) and MMTV-Cre;ppargf/f;mc4r−/−;Wnt (L) mammary
tumors is shown by H3K9me3 immunofluorescence microscopy. M Quantitation of LC3, Lamp1/Tom20, p16, and SAHF positive cells in mammary
tumors from both genotypes.

suggested that ketone bodies can induce cellular starvation
response and autophagy [34, 35]. Cytostatic autophagy was
shown to increase radiation sensitivity in lung cancer and breast
cancer cell lines [36–38]. These results indicate that autophagy is
an important cellular response to excess acetoacetate
concentration.
From a therapeutic standpoint, our results demonstrate that
HDAC3 inhibition induced autophagy and senescence in mouse
mammary tumors. HDAC3 expression is upregulated in breast
cancer and correlates with poor clinical outcomes [39]. HDAC3
overexpression promoted breast cancer cell proliferation and
aerobic glycolysis. The HDAC inhibitor vorinostat inhibited brain

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Fig. 3 Increased AASS expression and acetoacetate levels in mammary tumors from MMTV-Cre;ppargf/f;mc4r−/−;Wnt mice. A qRT-PCR
analysis of AASS expression in mammary tumors from mice with indicated genotypes. Error bars indicate SEM. AASS protein expression in
mammary tumors from MMTV-Cre;pparg+/+;mc4r−/−;Wnt (B) and MMTV-Cre;ppargf/f;mc4r−/−;Wnt (C) mice is shown by immunohis-
tochemistry. D Liver tissue was used as the positive control for AASS expression. Nuclei were counterstained with hematoxylin. Scale
bar = 10 μm. E Relative acetoacetate ion counts in mammary tumors from each genotype.

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Fig. 4 PPARγ repressor complex inhibits AASS gene activation in obese mammary tumors. A Relative PPRE binding by epigenetic repressor
proteins NCOR1, SMRT, and HDAC3 is shown by chromatin immunoprecipitation from MMTV-Cre;pparg+/+;mc4r−/−;Wnt and MMTV-
Cre;ppargf/f;mc4r−/−;Wnt mammary tumors. Relative PPRE occupancy by PPARγ and histone H3K27Ac is shown for each genotype. Pre-
immune IgG was used as the negative control. B Relative reporter gene activity is inhibited by the AASS PPRE as shown by luciferase analysis.
pGL3 reporter vectors were co-transfected with NCOR1, SMRT, HDAC3, CBP, PCAF, and PPARγ expression plasmids. The PPARγ synthetic ligand
rosiglitazone was used as a control for activation. The activities of luciferase reactions without reporter vector (blank) and PPRE mutant intron
is shown. Error bars indicate SEM. p values are shown.

metastasis in a triple negative breast cancer xenograft model
[40] but failed to induce ER expression [41]. HDAC inhibition
synergized with PARP inhibitors to induce synthetic lethality of
human triple negative breast cancer cell lines [42]. Vorinostat
synergized with histone demethylase inhibitors to induce
apoptosis in human triple negative breast cancer cell lines
[43]. Vorinostat also improved clinical responses in chemother-
apy treated breast cancer patients [44]. Inhibition of the sirtuin
family of HDACs induced autophagy in human breast cancer
cell lines [45]. These results indicate that HDAC inhibition is a
promising targeted therapy for breast cancer.
Our future studies will examine additional mechanisms by
which acetoacetate metabolism regulates autophagy in breast
cancer cells. We also will examine how the obese microenviron-
ment regulates PPARγ tumor suppressor function in mammary
cancer cells.
MATERIALS AND METHODS
Mouse breeding and procedures
Mouse strains and experimental procedures were approved by the
institutional animal care committee. The institution is accredited by AAALAC.
The Tg(MMTV-cre)1Mam/J, B6.129-Ppargtm2Rev/J, C57BL/6-Mc4rPkcp/JtJ, and
B6SJL-Tg(Wnt1)1Hev/J mutant mouse strains were purchased from The
Jackson Laboratory (Bar Harbor, ME, USA). The mouse mammary tumor virus
(MMTV) promoter directs Cre recombinase expression which introduces the

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Fig. 5 Decreased AASS expression in human breast cancer. Decreased AASS expression in human breast cancers (invasive ductal carcinoma,
IDC) and ductal carcinomas in situ (DCIS) compared to normal breast tissue from GEO datasets GDS3853 (A) and GDS2250 (B). C AASS
expression in molecular classifications of human breast cancer from TCGA was compared to normal breast tissue. D–I AASS protein expression
in representative samples of matched human breast tissue and cancers is shown by immunohistochemistry. Nuclei were counterstained with
hematoxylin. Scale bar = 10 μm. J AASS protein expression was determined in human breast cancer cell lines by western blot. Human
mammary epithelial cells (HMEC) extract was used as the positive control for AASS expression. β-actin expression was used to determine equal
amounts of protein in each lane. Representative blots are shown.

PPARγ null mutation in mammary epithelium. Mice were crossed to create
MMTV-Cre;pparg+/+;mc4r−/−;Wnt and MMTV-Cre;ppargf/f;mc4r−/−;Wnt
female offspring (n = 20 tumors/genotype; power = 0.8; alpha = 0.05;
S.D. = 1 based on tumor latency). Littermates were used to control for
genetic background and assigned to experimental or control groups by
genotype. In separate experiments, 20 tumor bearing MMTV-Cre;pparg
+/+;mc4r−/−;Wnt mice were treated with 100 mg/kg of the HDAC inhibitor
vorinostat or vehicle by oral gavage daily for 5 days. The latency, number,
and volume of tumors were recorded for each animal. Data were analyzed
by t-test. Mammary tumors were fixed in 4% buffered formaldehyde, flash
frozen and stored at −80 °C, or trypsin dissociated and cryopreserved in
liquid nitrogen. No animals or samples were excluded from analysis.
Investigators were blinded with respect to genotype. Statistical tests were
two sided.

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 J. Wu et al.
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Fig. 6 Reduced HDAC3 expression inhibits mammary tumorsphere proliferation and induces autophagy and senescence. A Relative
HDAC3 and AASS expression in tumor derived LP cells transduced with control or HDAC3 shRNAs is shown by qRT-PCR. Error bars indicate
SEM. p values are shown. In vitro tumorsphere formation by LP cells transduced with control (B) or HDAC3 (C) shRNAs is shown by phase
contrast microscopy. Scale bar = 50 μm. D Acetoacetate levels in LP derived tumorspheres transduced with control or HDAC3 shRNAs is
shown using a commercially available kit. E Expression of LC3 I and II in LP derived tumorspheres transduced with control or HDAC3 shRNAs is
shown by western blot. β-actin expression was used to determine equal amounts of protein in each lane. Cytoplasmic co-localization of
Lamp1+ lysosomes with Tom20+ mitochondria in LP derived tumorspheres transduced with control (F) and HDAC3 shRNAs (G) is shown by
immunofluorescence microscopy. Nuclei were counterstained with DAPI. Scale bar = 10 μm. H Quantitation of Lamp1+Tom20+ cells in both
treatment groups.

qRT-PCR Histopathology, immunofluorescence, and

RNA was extracted from mammary tumors and reverse transcribed according
to manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). cDNA was
amplified using mouse PPARγ primers 5′-AGCTGAATCACCCAGAGTCC-3′ and
5′-TGCAATCAATAGAAGGAACACG-3′, AASS 5′-GGGCTTACTGGGGGATGAAC-3′
and 5′-CTCGGGGAAAAGGAAGCACA-3′, HDAC3 5′-TTCCCTCAAACTCT-
CACGGC-3′ and 5′-CCCTCAGGTTCACCTTGCAT-3′. β-actin was amplified using
primers 5′-AAAAGCCACCCCCACTCCTAAG-3′ and 5'- TCAAGTCAGTGTA-
CAGGCCAGC-3′ at 94 °C for 25 s, 55 °C for 1 min, and 72 °C for 1 min.
Quantitative PCR was performed using the StepOnePlus system (Thermo
Fisher, Waltham, MA, USA) and normalized using the 2ΔΔCt method.
immunohistochemistry
Fixed mammary tumors (n = 20 per genotype) were dehydrated in
ethanol, cleared in xylene, and embedded in paraffin. Sections were
deparaffinized and stained with hematoxylin and eosin. For immunofluor-
escence analysis, sections were blocked using 10% normal serum and
incubated with Lamp1, Tom20, or H3K9me3 antibodies overnight at room
temperature. After washing, sections were incubated with secondary
antibodies conjugated to AlexaFluor 488 or AlexaFluor 555 at room
temperature for 1 h. After additional washing, sections were coverslipped
using anti-fade mounting medium containing DAPI and imaged using a

Oncogene (2023) 42:2402 – 2414

 J. Wu et al.
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Fig. 7 HDAC inhibition reduces cell proliferation and depletes the LP cell fraction in obese mammary tumors. The histopathologic
appearance of vehicle (A) and vorinostat (B) treated mc4r−/−;Wnt mammary tumors is shown by H&E staining. Scale bar = 10 μm. Cell
proliferation in vehicle (C) and vorinostat (D) treated mc4r−/−;Wnt mammary tumors is shown by PCNA immunohistochemistry. Nuclei were
counterstained with hematoxylin. E Quantitation of PCNA+ cells in each treatment group is shown. LP cell fraction in vehicle (F) and
vorinostat (G) treated mc4r−/−;Wnt mammary tumors is shown by flow cytometry. CD61 log scale and forward scatter linear scale is shown.
Quantitation of LP cells is shown. Programmed cell death in vehicle (H) and vorinostat (I) treated mc4r−/−;Wnt mammary tumors is shown by
TUNEL analysis. Nuclei were counterstained with DAPI. J Quantitation of TUNEL+ cells in vehicle and vorinostat treated mc4r−/−;Wnt
mammary tumors.

Zeiss 710 confocal microscope (White Plains, NY, USA). Human invasive
ductal breast cancer tissue microarrays were purchased from Super Bio
Chips, Seoul, Korea, US Biolab, Rockville, MD, and TissueArray, Rockville,
MD, USA (223 cases consisting of 138 estrogen receptor positive, 46
estrogen receptor negative, and 39 Her2 positive tumors). For immuno-
histochemical analysis, sections were blocked using 10% normal serum
and incubated with anti-PCNA, LC3, p16INK4A, or AASS antibodies overnight
at room temperature. After washing, sections were incubated with
biotinylated secondary antibody at room temperature for 10 min. After
additional washing, sections were incubated with streptavidin-conjugated
horseradish peroxidase for 10 min at room temperature. Antigen–antibody
complexes were detected by incubation with peroxide substrate solution
containing aminoethylcarbazole chromogen. Data were obtained using
the Leica Vectra 3 image analysis system and analyzed by t-test.
Cell death analysis
Mammary tumor sections (n = 20 per genotype) were incubated with
terminal deoxynucleotidyl transferase and dUTP-fluorescein for 1 h at 37 °C
according to manufacturer’s recommendations (Roche Applied Sciences,
Indianapolis, IN, USA). After washing, apoptotic cells were visualized by
fluorescence microscopy. The percent fluorescent cells was determined using
the Leica Vectra 3 image analysis system. Data were analyzed by t-test.

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 J. Wu et al.
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Fig. 8 The HDAC inhibitor vorinostat induces autophagy and senescence in mc4r−/−;Wnt mammary tumors. A Acetoacetate levels in
vehicle and vorinostat treated mc4r−/−;Wnt mammary tumors are shown using a commercially available kit. Expression of the autophagy
marker LC3 in vehicle (B) and vorinostat (C) treated mc4r−/−;Wnt mammary tumors is shown by immunohistochemistry. Nuclei were
counterstained with hematoxylin. Scale bar = 10 μm. Cytoplasmic co-localization of Lamp1+ lysosomes with Tom20+ mitochondria in vehicle
(D) and vorinostat (E) treated mc4r−/−;Wnt mammary tumors is shown by immunofluorescence microscopy. Nuclei were counterstained with
DAPI. Expression of the senescence marker p16INK4A in vehicle (F) and vorinostat (G) treated mc4r−/−;Wnt mammary tumors. Senescence
associated heterochromatin foci (SAHF) in vehicle (H) and vorinostat (I) treated mc4r−/−;Wnt mammary tumors. J Quantitation of LC3, Lamp1/
Tom20, p16, and SAHF positive cells in each treatment group is shown.

Transmission electron microscopy
Mammary tumors (n = 10 per genotype) were fixed in phosphate buffered
2% glutaraldehyde, postfixed in phosphate buffered 1% osmium tetroxide,
dehydrated in acetone series, infiltrated in propylene oxide/resin, and
embedded in Epon resin. In total, 0.1 μm sections were stained with uranyl
acetate and lead citrate and imaged using a JEM-1220 electron microscope
(JEOL, Peabody, MA, USA).
Western blot
Protein was extracted from mammary tumors or breast cancer cell lines in
1x Laemmli buffer. In total, 75 μg total cellular protein was separated by
SDS-PAGE. Proteins were electroblotted to PVDF membranes. Blots were
incubated with antibodies to LC3, AASS, pAkt, Akt, pAMPK, AMPK, Atg3,
Atg5, Atg6, Atg7, Atg12, Atg16, or β-actin for 16 h at 4 °C. After washing,
blots were incubated for 30 min at room temperature with anti-IgG
secondary antibody conjugated to horseradish peroxidase. Bands were
visualized by the enhanced chemiluminescence method, normalized to
β-actin expression, and quantitated using densitometric software (Silk
Scientific, Provo, UT, USA). Experiments were repeated three times. Data
were analyzed by ANOVA.
Fluorescence activated cell sorting
Mammary tumors (n = 20 per genotype; n = 20 for vehicle or vorinostat
treated MMTV-Cre;pparg+/+;mc4r−/−;Wnt groups) were dissociated by
trypsinization, washed in PBS, and incubated with phycoerythrin
conjugated CD61 antibody. Samples were washed in PBS followed by
FACS (Beckman Coulter, Indianapolis, IN, USA). Data were analyzed by t-
test.

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Tumorsphere culture and lentiviral transduction
Sorted CD61+ luminal progenitor cells from 10 mammary tumors were
suspension cultured in triplicate in stem cell medium (3:1 Dulbecco’s modified
Eagle medium/F12 medium, 1X B27 supplement, 10 ng/ml epidermal growth
factor, 25 ng/ml basic fibroblast growth factor, 0.2% heparin, 40 μg/ml
gentamicin, 2.5 μg/ml amphotericin B) for 7 days at 37 °C in a humidified
atmosphere of 5% CO2. Cells were incubated with HDAC3 shRNAs or control
lentiviruses with 5 μg/ml polybrene overnight according to manufacturer’s
protocol (Horizon Discovery, Lafayette, CO, USA) at 37 °C. MSC medium was
replaced and cells cultured for 24 h. Two μg/ml puromycin was added to
select for transduced cells. For tumorsphere analysis, clonogenicity, and
proliferation were determined by counting and tumorsphere diameter
measurements every second day for 2 weeks. Tumorspheres were photo-
graphed using an Olympus X51 inverted phase contrast microscope (Center
Valley, PA, USA). Data were analyzed by t-test.
Cell culture and transfection
Human breast cancer cell lines MCF7, T47D, MDA-MB-231, and MDA-MB-
468 were obtained from ATCC and cultured in triplicate in Dulbecco’s
modified Eagle medium, 10% fetal bovine serum, and 40 μg/ml gentamicin
at 37° C in a humidified atmosphere of 5% CO2. Cultures tested negative
for mycoplasma contamination. Cultures were treated with up to 1 mM
acetoacetate for 5 days. In separate experiments, cells were transfected
with 5 μg AASS expression plasmid (Addgene, Watertown, MA, USA) using
Lipofectamine and selected with 400 μg/ml G418 for 14 days. Proliferation
of stable clones was determined by daily counting of triplicate cultures.
Data were analyzed by t-test.
In vitro senescence analysis
Triplicate human breast cancer cell line cultures were fixed for 5 min in
phosphate buffered 2% formaldehyde and 0.2% glutaraldehyde at room
temperature. Cultures were incubated in 1 mg/ml X-gal solution containing
5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM
MgCl2, overnight at 37 °C and photographed by phase contrast
microscopy. Data were analyzed by t-test.
Chromatin immunoprecipitation
Mammary tumor LP cells were fixed in 1% formaldehyde for 10 min
followed by native lysis and DNA fragmentation. Purified chromatin was
immunoprecipitated using NCOR1, SMRT, HDAC3, H3K27Ac, or PPARγ
antibodies and protein A/G agarose beads. Eluted DNA fragments were
purified for PCR templates. The input fraction before immunoprecipitation
was amplified as the positive control. Control IgG immunoprecipitates
were used as the negative control. DNA templates were amplified with
primers 5′-ACTTCTGCTCTGACGTTGTCTT-3′ and 5′-GATACACATGCAAG-
CACTGAGAC-3′ flanking the PPARγ response element. Experiments were
repeated three times and data were analyzed by ANOVA.
Reporter gene analysis
A 1.2-kb fragment of intron 11 of the mouse AASS gene containing the
putative PPAR response element (5′- AGGTCAGAGGACA -3′) was cloned into
the pGL3 luciferase vector. We generated a mutant intron with a 3-bp
substitution in the putative PPARγ response element (5′-AGGcgcGAGGACA-3′)
using the QuikChange site directed mutagenesis kit (Promega, Fitchburg, WI,
USA). Wild-type and mutant constructs were confirmed by sequencing.
Reporter constructs were transfected with mouse PPARγ, NCOR1, SMRT,
HDAC3, CBP, or PCAF expression vectors or control plasmid and Renilla
luciferase reporter vector pRL-TK into the mouse mammary tumor cell line
NF639 using Lipofectamine. In separate experiments, cells were treated with
1 μM concentration of the PPARγ ligand rosiglitazone for 6 h. Reporter activity
was determined in triplicate using the dual luciferase assay kit (Promega,
Fitchburg, WI, USA) and luminometry (Berthold, Oak Ridge, TN, USA). Intron
activity was normalized to TK promoter activity. Experiments were repeated
three times and data were analyzed by ANOVA.
Acetoacetate analyses
Mammary tumor tissue (n = 5 from each genotype) was flash frozen in
liquid nitrogen, pulverized, and polar metabolites extracted in 2:1
methanol/water. Extracts were lyophilized and weighed for normalization
of polar metabolites. Aliquots of extracts were dissolved in acetonitrile with
1 mM hydrazinoquinoline and incubated at 60 °C for 1 h for acetoacetate
derivatization. Derivatized product was evaporated to dryness and
Oncogene (2023) 42:2402 – 2414
J. Wu et al.
2413
suspended in methanol. Acetoacetate was identified and quantitated
automatically using a QTRAP 6500 liquid chromatography-mass spectro-
meter (Sciex, Framingham, MA, USA) as relative ion counts using
manufacturer’s software, based on retention time and mass fragmentation
patterns matched against facility database and external standards.
Acetoacetate levels in primary mouse mammary tumor cells and human
breast cancer cell lines also were determined using a commercially
available kit (Millipore Sigma, MAK199, St. Louis, MO, USA). Data were
analyzed by t-test.
Statistical analysis
T test was used for parametric data in two groups. ANOVA was used for
multiple groups of parametric data. Tests were two sided. Fisher exact test
was used for nonparametric data. Data were normally distributed with
similar variance. Mean center values were used and error bars reflect
variation within groups of data (standard error of the mean).
Antibodies
Abcam H3K9me3 AB8898, H3K27Ac AB4729, HDAC3 AB137704, Lamp1
AB24170, LC3 AB48394, p16INK4A AB54210, Cell Signaling β-actin 4970S,
pAkt 9271T, Akt1 2938T, pAMPK 2535T, AMPK 5832T, Atg3 3415P, Atg5
12994P, Atg6 3495P, Atg7 8558P, Atg12 4180P, Atg16 8089P, Santa Cruz
AASS SC390536, CBP SC7300, CD61 SC19671, NCOR1 SC1609, PCAF
SC6300, PPARγ SC7196, SMRT SC1612, Tom20 SC11415, PCNA SC7907.
DATA AVAILABILITY
Data are available on reasonable request from the authors.
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ACKNOWLEDGEMENTS
We thank Drs. Ke Ma, Balaji Ganesh, Hui Chen, and Feigen Seiler (University of Illinois
Research Resources Center) for assistance with confocal microscopy, flow cytometry,
mass spectrometry, and electron microscopy. This study was supported by
Department of Defense Breast Cancer Research Program Award W81XWH-20-1-0029.
AUTHOR CONTRIBUTIONS
JW and KK performed experiments and analyzed data. DLC supervised the project
and wrote the manuscript.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41388-023-02766-8.
Correspondence and requests for materials should be addressed to David L. Crowe.
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Oncogene (2023) 42:2402 – 2414