Is cancer a metabolic rebellion against host aging? In the quest for immortality, tumor cells try to save themselves by boosting mitochondrial metabolism

Article (PDF Available)inCell cycle (Georgetown, Tex.) 11(2):253-63 · January 2012with42 Reads
DOI: 10.4161/cc.11.2.19006 · Source: PubMed
Abstract
Aging drives large systemic reductions in oxidative mitochondrial function, shifting the entire body metabolically towards aerobic glycolysis, a.k.a, the Warburg effect. Aging is also one of the most significant risk factors for the development of human cancers, including breast tumors. How are these two findings connected? One simplistic idea is that cancer cells rebel against the aging process by increasing their capacity for oxidative mitochondrial metabolism (OXPHOS). Then, local and systemic aerobic glycolysis in the aging host would provide energy-rich mitochondrial fuels (such as L-lactate and ketones) to directly "fuel" tumor cell growth and metastasis. This would establish a type of parasite-host relationship or "two-compartment tumor metabolism", with glycolytic/oxidative metabolic-coupling. The cancer cells ("the seeds") would flourish in this nutrient-rich microenvironment ("the soil"), which has been fertilized by host aging. In this scenario, cancer cells are only trying to save themselves from the consequences of aging, by engineering a metabolic mutiny, through the amplification of mitochondrial metabolism. We discuss the recent findings of Drs. Ron DePinho (MD Anderson) and Craig Thomspson (Sloan-Kettering) that are also consistent with this new hypothesis, linking cancer progression with metabolic aging. Using data mining and bioinformatics approaches, we also provide key evidence of a role for PGC1a/NRF1 signaling in the pathogenesis of (1) two-compartment tumor metabolism, and (2) mitochondrial biogenesis in human breast cancer cells.

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Available from: Michael P Lisanti
www.landesbioscience.com Cell Cycle 253
Cell Cycle 11:2, 253-263; January 15, 2012; © 2012 Landes Bioscience
PERSPECTIVE PERSPECTIVE
Key words: aging, mitochondria, cancer
metabolism, autophagy, mitophagy,
aerobic glycolysis, oxidative phosphoryla-
tion, Metformin, drug resistance,
chemoresistance, Warburg effect, meta-
bolic compartments, parasite, PGC1a,
PGC1b, NRF1, two-compartment tumor
metabolism
Submitted: 12/08/11
Accept e d : 12 / 25/11
http://dx.doi.org/10.4161/cc.11.2.19006
*Correspondence to: Michael P. Lisanti and
Federica Sotgia; Email: michael.lisanti@kimmelcan-
cercenter.org and federica.sotgia@jeerson.edu
Aging drives large systemic reduc-
tions in oxidative mitochondrial
function, shifting the entire body met-
abolically toward aerobic glycolysis,
a.k.a, the Warburg effect. Aging is also
one of the most significant risk factors
for the development of human cancers,
including breast tumors. How are these
two findings connected? One simplis-
tic idea is that cancer cells rebel against
the aging process by increasing their
capacity for oxidative mitochondrial
metabolism (OXPHOS). Then, local
and systemic aerobic glycolysis in the
aging host would provide energy-rich
mitochondrial fuels (such as L-lactate
and ketones) to directly “fuel” tumor
cell growth and metastasis. This would
establish a type of parasite-host rela-
tionship or “two-compartment tumor
metabolism,” with glycolytic/oxidative
metabolic coupling. The cancer cells
(“the seeds”) would flourish in this
nutrient-rich microenvironment (“the
soil”), which has been fertilized by host
aging. In this scenario, cancer cells are
only trying to save themselves from the
consequences of aging by engineering a
metabolic mutiny, through the amplifi-
cation of mitochondrial metabolism. We
discuss the recent findings of Drs. Ron
DePinho (MD Anderson) and Craig
Thomspson (Sloan-Kettering) that are
also consistent with this new hypoth-
esis, linking cancer progression with
metabolic aging. Using data mining and
Is cancer a metabolic rebellion against host aging?
In the quest for immortality, tumor cells try to save themselves by boosting
mitochondrial metabolism
Adam Ertel,1,2 Aristotelis Tsirigos,3 Diana Whitaker-Menezes,1,2 Ruth C. Birbe,4 Stephanos Pavlides,1,2,5 Ubaldo E. Martinez-
Outschoorn,1,2, 6 Richard G. Pestell,1,2 ,6 Anthony Howell,5 Federica Sotgia1,2,5,* and Michael P. Lisanti1,2 ,5,6,*
1The Jefferson Stem Cell Biology and Regenerative Medicine Center; 2Departments of Stem Cell Biology and Regenerative Medicine and Cancer Biology;
4Department of Pathology, Anatomy & Cell Biology; 6Department of Medical Oncology; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, PA
USA; 3Computational Genomics Group; IBM Thomas J. Watson Research Center; Yorktown Heights, NY USA; 5Manchester Breast Centre & Breakthrough
Breast Cancer Research Unit; Paterson Institute for Cancer Research; Manchester, UK
bioinformatics approaches, we also pro-
vide key evidence of a role for PGC1a/
NRF1 signaling in the pathogenesis of
(1) two-compartment tumor metabo-
lism and (2) mitochondrial biogenesis in
human breast cancer cells.
Aging, Metabolic Decline
and Cancer
During aging, oxidative stress and reac-
tive oxygen species (ROS) lead to accu-
mulated DNA damage and mitochondrial
dysfunction.1-10 As a consequence, dra-
matic reductions in oxygen consumption
occur, shifting the entire body toward
glycolytic metabolism or aerobic glycoly-
sis11-13 (Fig. 1). This produces energy-
rich metabolites, such as L-lactate, as a
by-product.14-16 In fact, high systemic
and local L-lactate levels are considered
as a hallmark of aging.14-16 As such, the
morbidity and mortality associated with
aging could be mediated, in part, by the
metabolic collapse of oxidative mitochon-
drial metabolism. If organismal death is
mediated by mitochondrial dysfunction
and metabolic catastrophe, then organ-
ismal longevity or immortality could be
achieved by mitochondrial rejuvenation or
amplification. Interestingly, immortality
is one of the hallmarks of cancer cells.
Remarkably, aging is also one of the
single most important risk factors associ-
ated with cancer (See http://info.cancerre-
searchuk.org/cancerstats/incidence/age/).
254 Cell Cycle Volume 11 Issue 2
would set up a two-compartment meta-
bolic system, in which the tumor stroma
(aging host) is glycolytic and the cancer
cells are oxidative (Figs. 2 and 3). In this
two-compartment system, oxidative can-
cer cells and glycolytic host cells would be
metabolically coupled in a type of host-
parasite relationship. Then, tumor cells
would directly “feed” off the aging host
microenvironment, much like an infec-
tious parasite.17-23
In fact, canine transmissible vene-
real tumor (CTVT) is an example of an
infectious “parasitic” cancer cell that is
metastatically transmitted from one host
dog to another by allografting during
copulation.24,25 Via molecular analysis
of its nuclear genomic DNA, it has been
estimated that this cancer cell originated
> 10,000 years ago from a coyote or wolf-
like animal and is now serially passed from
one dog to another as a “parasitic organ-
ism.”24 As such, this is the oldest known
cancer cell line that has been continuously
propagated.24
How did this cancer cell achieve
immortality? The answer comes from
analysis of its mitochondrial DNA
(mtDNA), which shows that it is derived
from the modern dog (Canis lupus famil-
iaris), its current host.26 Thus, CTVT
cells “steal” host mtDNA, allowing for
mitochondrial amplification and reju-
venation. Mechanistically, others have
shown that mesenchymal stem cells
(MSCs) and fibroblasts can also actively
transfer intact mitochondria via nano-
tubes to epithelial cancer cells, allow-
ing for mitochondrial rejuvenation and
reversal of aerobic glycolysis.27 This is
known simply as mitochondrial trans-
fer.27 Similar results have also been now
obtained using B16 melanoma cells and
4T1 mammary cancer cells after implan-
tation in murine hosts, which obtain host
mtDNA, most likely via a similar mecha-
nism.28 Thus, cancer cells can effectively
steal host cell mitochondria and mtDNA
to increase oxidative mitochondrial
metabolism.
In accordance with the above observa-
tions, we have recently shown that epi-
thelial cancer cells behave as metabolic
parasites, using a co-culture approach
employing human fibroblasts and MCF7
breast cancer cells29 (Fig. 3). In this
by bolstering mitochondrial function. Is
cancer simply a metabolic mutiny against
aging?
Aging Promotes
Two-Compartment Tumor
Metabolism: Mechanistic and
Therapeutic Implications
If cancer cells developed increased oxi-
dative mitochondrial function, then this
Nearly 65% of cancers occur in patients
≥65 years old, and more than a third are
detected in patients that are ≥ 75 years
old. Is there a metabolic connection here?
One testable hypothesis is that cancer cells
attain a growth advantage in the aging
host by increasing or amplifying their oxi-
dative capacity, through enhanced mito-
chondrial biogenesis (Fig. 2). While the
entire body is undergoing metabolic col-
lapse, cancer cells try to save themselves
Figure 1. Aging induces whole-body aerobic glycolysis. In humans, several independent physio-
logic studies have shown that oxygen consumption steadily declines with aging (red line), shifting
the entire body toward glycolytic metabolism, even in the presence of oxygen. As a consequence,
aging and mortality are associated with aerobic glycolysis, while longevit y and immortality would
be associated with oxidative mitochondrial metabolism. Thus, the successful maintenance of mito-
chondrial “health” may be a key determinant of the life and the death of an organism.
Figure 2. Power surge: Cancer cells rebel against metabolic aging. Metabolic aging is charac-
terized by a shift toward aerobic glycolysis. Thus, the whole body shows a decrease in ox ygen
consumption. In order to save themselves from aging-induced metabolic catastrophe, cancer
cells could increase their capacity for oxidative mitochondrial metabolism (up arrow). This would
set up two distinct metabolic compartments within a tumor: glycolytic and oxidative. Aerobic
glycolysis in the tumor stroma (the host) could provide energy-rich nutrients (down arrow). Then,
oxidative tumor cells could use these nutrients to fuel oxidative mitochondrial metabolism, gen-
erating a type of host-parasite relationship or metabolic coupling.
www.landesbioscience.com Cell Cycle 255
Visualizing Two-Compartment
Tumor Metabolism:
Hyperactivation of Mitochondrial
Metabolism in Cancer Cells
To test the hypothesis that the tumor
stroma is glycolytic and that epithelial
cancer cells are oxidative in vivo, we used
an established method to detect func-
tional mitochondrial activity in frozen
sections.72 This method has been suc-
cessfully used for the past 50 years to
diagnose mitochondrial disease in clini-
cal muscle biopsies, as skeletal muscle is
a mitochondria-rich organ system.72 It is
the “seed and soil” hypothesis67-70 first
proposed in 1889 by Dr. Stephen Paget
(Fig. 5). In this hypothesis, cancer cells
(the seeds) grow and propagate best in
right mircroenvironment (the soil).6 8-70
In this context, the aging host would pro-
vide an exceptionally “fertile ground” for
parasitic cancer cells with increased mito-
chondrial power via the production of
host-derived mitochondrial fuels, such as
L-lactate, ketones and glutamine as well as
free fatty acids.23,71 Thus, aging-induced
metabolic decline may be functionally fer-
tilizing the soil for cancer cell growth and
metastasis.
co-culture system, we observed that a
two-compartment metabolic system was
established. Cancer cells first secreted
hydrogen peroxide (H2O2) to induce
oxidative stress in adjacent fibroblasts as
a form of accelerated aging;30-32 at the
same time, the cancer cells mounted an
antioxidant defense by upregulating anti-
oxidant proteins, such as TIGAR and
peroxiredoxins.29,33,34
Oxidative stress in the cancer-associ-
ated fibroblasts increased stromal ROS
production, activating two major tran-
scription factors, namely, HIF1a and
NFκB, which both function as master reg-
ulators of autophagy, mitophagy, aerobic
glycolysis as well as inflammation.30,33 -36
As a consequence, the stromal fibro-
blasts would produce high-energy
nutrients (L-lactate, ketones and glu-
tamine).22, 37-40 In turn, these nutrients
stimulated mitochondrial biogenesis,
OXPHOS and autophagy resistance in
the epithelial cancer cells and protected
the cancer cells against basal and chemo-
therapy-induced apoptosis.33 ,34 ,41- 45 Thus,
two-compartment tumor metabolism and
mitochondrial “health” may be the basis
of chemoresistance and therapy failure in
cancer patients.23,46
Given that two-compartment tumor
metabolism may be a clinical barrier to
effective cancer treatments, normalizing
energy balance (homeostasis) should cut
off the fuel supply to cancer cell mito-
chondria (Fig. 4). Drugs such as NAC
(N-acetyl-cysteine) and chloroquine will
inhibit oxidative stress and autophagy in
the tumor stroma, preventing the produc-
tion of high-energy mitochondrial fuels.
Conversely, Metformin (a mitochondrial
complex I inhibitor)47 should prevent
cancer cells from effectively using mito-
chondrial fuels by inhibiting OXPHOS
in cancer cells, inducing the conventional
Warburg effect in cancer cells.48 -52 In fact,
all three drugs have been shown to func-
tion as anticancer agents and also are asso-
ciated with increased lifespan in studies
of longevity.53 -66 So, a global whole-body
approach to normalizing energy bal-
ance may prevent both aging and cancer,
simultaneously.
This discussion of two-compartment
tumor metabolism is also reminiscent of
Figure 3. Understanding two-compartment tumor metabolism. In two-compartment tumor
metabolism, the stroma and the tumor cells have opposite metabolic phenotypes, allowing for
metabolic coupling. Thus, the tumor stroma (and the aging host) would be characterized by oxi-
dative stress, autophagy, mitophagy and aerobic glycolysis. In contrast, cancer cells would mount
an antioxidant defense, become autophagy-resistant, increase mitochondrial biogenesis and
undergo oxidative phosphorylation. Aging naturally induces oxidative stress. Oxidative stress, in
turn, is sucient to drive autophagy, mitophagy and aerobic glycolysis. Tumor cells can protec t
themselves against all of these catabolic processes by mounting an antioxidant defense by delet-
ing autophagy genes and/or by increasing their mitochondrial mass.
256 Cell Cycle Volume 11 Issue 2
of two-compartment tumor metabo-
lism.72 Epithelial cancer cell nests are
heavily stained and are COX-positive.
In striking contrast, the tumor stroma is
COX-negative.72 These observations are
consistent with the idea that epithelial
cancer cells use oxidative mitochondrial
metabolism, while the tumor stromal cells
are largely glycolytic by comparison.72
Importantly, normal adjacent epithelial
cells show much less COX activity than
cancer cells (Fig. 6, see inset). It appears
that oxidative mitochondrial metabolism
is selectively amplified or hyperactivated
in cancer cells.72 Thus, cancer cells do
indeed appear to successfully rebel against
the metabolic decline observed during
aging.
Virtually identical results were
obtained with other mitochondrial stains
that detect t he functional activities of com-
plex I and II, highlighting the generality of
these findings.72 Positive staining was also
abolished using mitochondrial inhibitors,
such as Metformin, a complex I inhibi-
tor.72 Thus, inhibition of mitochondrial
function may mechanistically explain why
Metformin increases organismal longevity
and prevents a host of different types of
cancers in diabetic patients.
To independently validate these
observations, we used a bioinformatics
approach to re-analyze the transcriptional
profiles of epithelial cancer cells and adja-
cent stromal cells that were separated by
laser capture microdissection from n = 28
human breast cancer patients.72 As shown
in Figure 7, key components of mito-
chondrial complexes I–V were all tran-
scriptionally upregulated in human breast
cancer epithelial cells and, hence, down-
regulated in adjacent stromal cells. Other
mitochondrial-associated genes involved
in the TCA cycle and mitochondrial
protein translation were also upregulated
selectively in epithelial cancer cells.72 This
transcriptional evidence independently
supports our functional results from
COX-activity staining.72
Overexpression of this epithelial-spe-
cific MITO/OXPHOS gene signature was
observed in a majority of human breast
cancer patients (> 2,000 examined, with
a p-value < 10-20 ) and was specifically
associated with metastasis, especially in
ER-negative breast cancer patients.72
respiratory enzyme,” now known as
complex IV (www.nobelprize.org/
nobel_prizes/medicine/laureates/1931/
warburg-bio.html).
Figure 6 shows that COX stain-
ing of human breast cancer frozen sec-
tions allows the direction visualization
known as COX (cytochrome C oxidase)-
staining and detects the functional activ-
ity of mitochondrial complex IV, one
of the last steps in oxidative mitochon-
drial metabolism (OXPHOS). In fact, in
1931, Dr. Otto Warburg won the Nobel
prize for the discovery of the “Warburg
Figure 4. Restoring energy balance: Preventing two-compartment tumor metabolism. In the
tumor stroma (and the aging host), oxidative stress activates two major transcription factors,
namely, HIF1α and NFκB. Both are positive regulators of autophagy, mitophagy and aerobic gly-
colysis as well as the inammatory response. In contrast, cancer cells could increase mitochondrial
biogenesis via the activation of key transcription factors, such as PGC1a and NRF1. Thus, targeted
therapies should reduce oxidative stress (NAC, N-acetyl-cysteine) or autophagy (chloroquine) in
the tumor stroma (aging host). In contrast, tumor cell mitochondria could be targeted with mito-
chondrial inhibitors, such as Met formin. Both approaches would shut-down two -compartment
tumor metabolism by normalizing metabolism or restoring homeostasis/energy balance.
Figure 5. The seed and soil hypothesis and the aging host. In the seed and soil hypothesis, rst
proposed by Stephen Paget in 1889, cancer cells (“the seeds”) grow best in the most fertile tumor
microenvironment (“the soil” ). In this context, oxidative cancer cells would grow best surrounded
by glycolytic host cells, which would provide a metabolic engine for tumor growth and metasta-
sis. Such parasitic metabolic coupling would be consistent with the seed and soil hypothesis.
www.landesbioscience.com Cell Cycle 257
Data Mining and Bioinformatics
Analysis of PGC1a/NRF1-
Signaling in Human Breast
Cancers: Association
with Metastasis, Recurrence
and Poor Overall Survival
Here, to determine if PGC1a and NRF1
(nuclear respiratory factor 1) activity is
upregulated in human breast cancers, we
performed a bioinformatics analysis using
existing published data sets. For this pur-
pose, we re-interrogated a set of n = 28
human breast cancer patient samples83 in
which the tu mor stroma and epithelial ca n-
cer cells were physically separated by laser
recent study directly showed that targeted
deletion of PGC1a in mice prevents car-
cinogen-induced epithelial tumorigenesis
in the liver and the colon.78 Conversely,
overexpression of PGC1a in cancer cells
dramatically increases tumor growth in
murine animal models.78 Similarly, silenc-
ing of PGC1a also suppresses the growth
of AR-expressing prostate cancer cells.79 In
addition, the PGC1a pathway and mito-
chondrial biogenesis appear to be upregu-
lated or activated in endometrial cancers.80
In accordance with an overall dependence
of cancer cells on mitochondrial biogen-
esis, inhibition of mitochondrial transla-
tion blocks tumor growth.81, 82
A New Theory for Aging that
Connects Telomerase with
Mitochondrial Function
Recently, Dr. Ron DePinho
(MD Anderson/Dana Farber) proposed a
new theory for aging based on his experi-
ments with telomerase-deficient mice73-76
(Fig. 8). More specifically, his laboratory
showed that telomerase-deficient mice
show signs of accelerated aging and met-
abolic dysfunction with a loss of mito-
chondrial activity.73 He proposed that a
loss of telomerase activity during aging
induces activation of p53, which decreases
the expression and/or activity of certain
key target genes, such as PGC1a/b.73
Importantly, PGC1a/b functions as a
major positive transcriptional regulator of
mitochondrial genes, in part through the
activation of another downstream target,
namely NRF1 (nuclear respiratory fac-
tor 1). Thus, a loss of PGC1a/b results
in decreased mitochondrial function and
reduced OXPHOS and mitochondrial
stress.73 In further support of this hypoth-
esis, genetic ablation of p53 functionally
reverted this accelerated aging pheno-
type, restoring normal mitochondrial
function.73
Although DePinho’s group did not
specifically explore the role of this pos-
sible mechanism in cancer cells, it is rela-
tively straightforward to postulate that
just the opposite signaling pathway may
be operating in tumor cells. As such,
increased telomerase function and the
deletion of p53, which is thought to occur
in a majority of human cancer cell types,
would be predicted to increase PGC1a/b
activity and boost mitochondrial func-
tion (Fig. 8). This would provide another
possible mechanism by which cancer cells
could rebel against or escape from meta-
bolic aging and increase their oxidative
mitochondrial power, avoiding metabolic
decline.
Is there any experimental evidence to
support this hypothesis? For example,
consistent with this hypothesis, Craig
Thomspon and colleagues (Sloan-
Kettering/UPENN) have shown that
Metformin, a mitochondrial poison, is
most beneficial in the treatment of p53(-/-)
tumor cell xenografts, as compared with
p53(+/+) cancer cells.77 Furthermore, a
Figure 6. Visualizing two-compartment tumor metabolism with mitochondrial activity
staining. Frozen sections from human breast cancers were subjected to COX staining (brown
color), which functionally detec ts mitochondrial activit y. Note that epithelial cancer cell nests
are COX-positive and, thus, are oxidative. In contrast, the tumor stroma is COX-negative and,
hence, glycolytic. Inset, “normal” adjacent epithelial cells (red arrow) show substantially less
COX activity as compared with epithelial cancer cells. Reproduced and modied with permis-
sion from reference 72.
258 Cell Cycle Volume 11 Issue 2
tumor metabolism and (2) mitochondrial
biogenesis in human breast cancer cells.
To further validate the idea that NRF1
expression is selectively upregulated in
human breast cancer cells relative to
adjacent stromal tissue, we performed
immunostaining with antibodies directed
against NRF1. Figures 14 and 15 directly
show that NRF1 protein expression is
largely confined to epithelial cancer cell
nests and preferentially excluded from
adjacent stromal cells, as predicted based
on our informatics analysis.
Summary and Conclusions
In summary, we propose that cancer cells
arise as a metabolic rebellion against host
aging. In the quest for immortality, cancer
cells hyperactivate or amplify oxidative
mitochondrial metabolism to escape from
aging-induced metabolic decline (aerobic
glycolysis). This sets up two-compartment
tumor metabolism, where cancer cells are
oxidative and the tumor stroma (the aging
host) is glycolytic, resulting in a “para-
sitic” form of energy transfer. Activation
of oxidative mitochondrial metabolism
in epithelial cancer cells may occur by
various mechanism(s) but likely involves
increased mitochondrial biogenesis via
PGC1/NRF1 signaling. Thus, we should
clinically target two-compartment tumor
metabolism and cancer cell mitochondria
to prevent and treat human cancers. The
drugs that would be developed may also
have beneficial side effects, as they should
also function as anti-aging therapies and
increase organismal longevity.
Acknowledgements
F.S. and her laboratory were supported by
grants from the Breast Cancer Alliance
(BCA) and the American Cancer Society
(ACS). U.E.M. was supported by a Young
Investigator Award from the Margaret
Q. Landenberger Research Foundation.
M.P.L. was supported by grants from
the NIH/NCI (R01-CA-080250;
R01-CA-098779; R01-CA-120876;
R01-AR-055660), and the Susan G.
Komen Breast Cancer Foundation. R.G.P.
was supported by grants from the NIH/
NCI (R01-CA-70896, R01-CA-75503,
R01-CA-86072 and R01-CA-107382)
and the Dr. Ralph and Marian C. Falk
tumors (> 2,000 cases examined) relative
to normal healthy breast tissue (102 con-
trols). Similarly, significant associations
were obtained with tumor tissue from both
ER-positive (> 1,600 cases examined) and
ER-negative (nearly 500 cases examined)
breast cancer patients. As such, upregula-
tion of PGC1/NRF1 signaling may be a
common feature of human breast cancers.
By employing the transcriptional sig-
nature for NRF1 target genes, we also
performed survival analysis using existing
transcriptional profiling data and acces-
sible outcome data from human breast
cancer patients. Fi g u res 11–13 show that
that when NRF1 target genes are tran-
scriptionally upregulated in human breast
cancers, there is a specific association with
metastasis, recurrence and poor overall
survival, especially in ER+/Luminal A
breast cancer patients.
Thus, this informatics analysis pro-
vides suggestive evidence of an impor-
tant role for PGC1/NRF1 signaling in
the pathogenesis of (1) two-compartment
capture microdissection (LSCM). The
transcriptional profiles of these two cel-
lular compartments (epithelia vs. stroma)
were used to generate a list of genes that
were specifically upregulated in epithe-
lial cancer cells, relative to adjacent stro-
mal tissue (2,901 transcripts upregulated
≥2-fold; p < 0.05). Then, this breast can-
cer gene set was intersected with the corre-
sponding gene sets for PGC1a and NRF1
target genes, obtained from the Molecular
Signatures Database (MSigDB).
Figure 9 shows two sets of Venn dia-
grams that summarize the results of this
data-mining analysis. Note that both
PGC1a and NRF1 target genes are both
upregulated specifically in human breast
cancer epithelial cells relative to adja-
cent stromal cells, with p-values between
1 x 10-10 and 10-21. Gene lists containing
these intersecting gene sets are included as
Tables S1 and S2.
Importantly, Figu re 10 shows that
the signatures for PGC1a and NRF1 are
also clearly upregulated in human breast
Figure 7. Mitochondrial genes are selectively upregulated in human breast cancer epithelial cells.
Using a bio-informatics approach, we re-analyzed the transcriptional proles of epithelial cancer
cells and adjacent stromal tissue, which were separated by laser-capture microdissection (from
n = 28 human breast cancer patients). Note that mitochondrial genes encoding subunits of com-
plexes I–V, which perform oxidative phosphorylation are upregulated >4-fold in epithelial cancer
cells as compared with adjacent stromal tissue. Only transcripts showing a >4-fold increase were
selected for the analysis, so this probably underestimates the overall increase in mitochondrial
OXPHOS capacity.72
www.landesbioscience.com Cell Cycle 259
Figure 9. Venn diagrams supporting a role
for PGC1/NRF signaling in human breast
cancers. To determine if PGC1a/NRF1 activity
is upregulated in human breast cancers, we
performed a bioinformatics analysis. Briey,
we re-interrogated a set of n = 28 human
breast cancer patient samples83 in which the
tumor stroma and epithelial cancer cells were
separated by laser-capture microdissection.
The transcriptional proles of these two cellu-
lar compartments (epithelia vs. stroma) were
used to generate a list of genes that were
upregulated in epithelial cancer cells, relative
to adjacent stromal tissue (2,901 transcripts
upregulated >2-fold; p < 0.05). Then, this
breast cancer gene set was intersected with
the corresponding gene sets for PGC1a and
NRF1 target genes obtained from the MSigDB
(MOOTHA_PGC and RCGCANGCGY_V$NRF1_
Q6). See also the following web links: w ww.
broadinstitute.org/gsea/msigdb/cards/PGC.
html and www.broadinstitute.org/gsea/msig-
db/cards/RCGCANGCGY_V$NRF1_Q6.html
Figure 8. Aging connects telomerase with mitochondrial function. Dr. Ron DePinho’s labora-
tory showed that telomerase -decient mice show signs of accelerated aging and metabolic
dysfunction, with a loss of mitochondrial ac tivity. He proposed that a loss of telomerase activity
during aging induces activation of p53, which decreases the expression and/or activity of certain
target genes, such as PGC1a/b. PGC1a/b functions as a major positive transcriptional regulator of
nucleus-encoded mitochondrial genes. Thus, a loss of PGC1a/b results in decreased mitochondrial
function and reduced OXPHOS and mitochondrial stress. Conversely, we speculate here that in-
creased telomerase function and the deletion of p53, which occurs in a majority of human cancer
cell types, would be predicted to increase PGC1a/b activity and boost mitochondrial func tion.
This could drive the onset of two-compartment tumor metabolism.
Medical Research Trust. The Kimmel
Cancer Center was supported by the
NIH/NCI Cancer Center Core grant
P30-CA-56036 (to R.G.P.). Funds were
also contributed by the Margaret Q.
Landenberger Research Foundation (to
M.P.L.). This project is funded, in part,
under a grant with the Pennsylvania
Department of Health (to M.P.L.
and F.S.). The Department specifically
disclaims responsibility for any analyses,
interpretations or conclusions. This work
was also supported, in part, by a Centre
grant in Manchester from Breakthrough
Breast Cancer in the UK (to A.H.) and an
Advanced ERC Grant from the European
Research Council.
Note
Supplemental material can be found at:
www.landesbioscience.com/journals/cc/
article /1900 6 /
References
1. Blagos klonny MV. Cell im mortality a nd hallma rks of
cancer. Cell Cycle 2003; 2:296-9; PMID:12851477;
http://dx.doi.org/10.4161/cc.2.4.470.
2. Blagosk lonny MV. Paradoxes of aging. Cell Cycle
2007; 6:2997-3003 ; PMID :18156807; http://dx.doi.
org/10.4161/cc.6.24.5124.
3. Blag osklonny MV. Progra m-like agi ng and mitochon-
dria: instead of random damage by free radicals. J
Cell Biochem 2007; 102:1389-99 ; PMID:17975792;
htt p: //dx .doi.org/10.1002/jcb.21602.
4. Blagosklonny MV. Prevention of cancer by inhib-
iting aging. Cancer Biol Ther 2008; 7:1520-4 ;
PMI D:18769112; ht tp ://d x.doi.org /10.4161/
cbt.7.10.6663.
5. Blagosklonny M V. Validation of anti-aging drugs
by treating age-related disea ses. A ging (Albany NY )
2009 ; 1:281-8; PMID :20157517.
6. Blagosklonny MV. TOR-driven aging: speeding
car wit hout brakes. Cell Cycle 2009; 8 :4055-9;
PMID:19923900; http://dx.doi.org/10.4161/
cc.8. 24.10310.
7. Blagosklonny MV. Aging-suppressants : cellu-
lar senescence (hyperactivation) and its pharma-
cologic deceleration. Cell Cycle 2009; 8:1883-7;
PMID:19448395; http://dx.doi.org/10.4161/
cc.8.12.8815.
8. Blagosklonny M V, Campisi J. Ca ncer and aging:
more puzzles, more promises ? Cell Cycle 2008;
7:2615-8; PMID :18719390; http://d x.doi.
or g / 10 . 41 61 / c c .7. 17. 6 6 2 6.
9. Bla gosklonny MV, Campisi J, Sinclair DA. A ging :
past, present and f uture. Aging (Albany NY ) 2009;
1:1-5 ; PMID :2 0157590.
10. Bla gosklonny MV, Hall MN. Growth and aging: a
common molecular mechanism. Aging (Albany NY)
2009 ; 1:357-62; PMID:20157523.
11. Fleg JL , Morrell CH, Bos AG, Brant LJ, Talbot LA,
Wright JG, et al. Accelerated longitudinal decline
of aerobic capacity in healthyears older adults.
Circulation 2005; 112: 674-82; PMID:16043637
260 Cell Cycle Volume 11 Issue 2
12. Hollenberg M, Yang J, Haight TJ, Tager IB.
Longitudina l changes in aerobic capacity: implica-
tions for concepts of a ging. J Gerontol A Biol Sci Med
Sci 2006; 61:851-8; PMID:16912104; http://dx.doi.
org/10.1093/gerona/61.8.851.
13. Tanaka H, Desouza CA, Jones PP, Stevenson ET,
Davy K P, Seals DR. Greater rate of decline in ma xi-
mal aerobic capacity with age in physically active
vs. sedentary healthy women. J Appl Physiol 1997;
83:1947-53; PMID:9390967.
14. Ross JM, Oberg J, Brene S, Coppotelli G, Terzioglu
M, Pernold K, et a l. High brain lactate is a hallmark
of aging and caused by a shift in the lactate dehy-
drogenase A/B ratio. Proc Natl Acad Sci USA 2010;
107:20087-92; PMID:21041631; http://dx.doi.
org /10.1073/pna s.10 08189107.
15. Bittles A H, Harper N. Increased glycolysis in aging
cultured human diploid f ibroblasts. Biosci Rep 1984;
4: 751-6; P MID: 6509159; http ://d x.doi. org/10.10 07/
BF01128816.
16. Goldstein S, Ballantyne SR, Robson AL , Moerman
EJ. Energy metabolism in cultured human fibrobl asts
during a ging in vitro. J Cell Physiol 1982; 112:419-
24; PMID:6127343; http://dx.doi.org/10.1002/
jcp.1041120316 .
17. Sotgia F, Martinez-Outschoorn U E, Howell A,
Pestell RG, Pavlides S, Lisanti MP. Caveolin-1 and
Cancer Metabolism in Tumor Progression: Markers,
Models and Mechanisms. Annu Rev Pathol 2012;
7: 4 2 3 - 6 7.
18. Sotgia F, Martinez-Outschoorn UE, Lisanti MP.
Mitochondrial oxidative stress drives tumor progres-
sion and metastasis: should we use antioxidants as a
key component of cancer treatment and prevention?
BMC Med 2011; 9:62 ; PMID :21605374; http ://
dx .d oi .org /10 .1186/1741-7015-9 - 62 .
19. Sotgia F, Mar tinez-Outschoorn UE, Pavlides S,
Howell A, Pestell RG, Lisanti MP. Understanding
the Warburg effect and the prognostic value of
stromal caveolin-1 as a marker of a lethal tumor
microenvironment. Breast Cancer Res 2011; 13:213;
PMI D:218 67571; htt p://dx.doi .org/10.1186/
bcr2892.
20. Martinez-Outschoorn UE, Pavlides S, Howell A,
Pestell RG, Tanowitz HB, Sotgia F, et al. Stromal-
epithelial metabolic coupling in cancer: integrating
autophag y and metabolism in the tumor microen-
vironment. Int J Biochem Cell Biol 2011; 43:1045-
51; PMID:21300172; http://dx.doi.org/10.1016/j.
biocel.2011.01.023.
21. Martinez-Outschoorn UE , Whitaker-Menezes D,
Pavlides S, Chiavarina B, Bonuccelli G, Casey T, et
al. The autophagic tumor stroma model of cancer or
“battery-operated tumor growth”: A simple solution
to the autophagy paradox. Cell Cycle 2010; 9: 4297-
306 ; PMID: 21051947; http ://dx.d oi.org /10.4161/
cc.9.21.13817.
22. Pavlides S, Vera I, Gandara R, Sneddon S, Pestell R,
Mercier I, et a l. Warburg Meets Autophagy: Cancer
Associated Fibroblasts Accelerate Tumor Growth
and Metastasis Via Oxidative Stress, Mitophagy and
Aerobic Glycolysis. Antioxid Redox Signal 2011; In
press; PMID:21883043.
23. Martinez-Outschoorn UE, Pestell RG, Howell A,
Nagajyothi F, Machado FS, Tanowitz HB, et al.
Energy transfer in “parasitic” cancer metabolism:
Mitochondria are the powerhouse and Achilles’
heel of tumor cells. Cell Cycle 2011; 10:4208-16;
PMID :22033146.
24. Rebbeck CA, Thomas R, Breen M, Leroi AM, Burt
A. Origins and evolution of a transmissible cancer.
Evolution 20 09; 63: 2340-9 ; PMID:19453727; http ://
dx .doi.or g/10 .1111/ j.1558 -5 646 .20 09.00 72 4.x .
25. Murchison EP. Clonally transmissible ca ncers in
dogs and Tasmanian devils. Oncogene 2008; 27:19-
30; PMID:19956175; http://dx.doi.org/10.1038/
onc.2009.350.
Figure 10. PGC1a and NRF1 target genes are transcriptionally upregulated in human breast
cancers. Note that the signatures for PGC1a and NRF1 target genes are upregulated in human
breast tumors (>2,000 cases examined), relative to normal healthy breast tissue (102 controls). In
addition, signicant associations were obtained with tumor tissue from both ER-positive (>1,600
cases examined) and ER-negative (nearly 500 cases examined) breast cancer patients. Thus, up -
regulation of PGC1a/NRF1 target genes is a general feature of human breast cancers. p -values are
as indicated. Dierential expression of the averaged gene signature magnitude between sample
groups was evaluated using a two-tailed t-test. This informatics analysis was performed essen-
tially as we previously described in references 72 and 84.
Figure 11. Kaplan-Meier analysis of NRF1 target genes in breast cancer metastasis. Note that the
signature for NRF1 target genes predicts breast cancer cell metastasis, especially in ER-positive
patients. Numbers of cases with annotation are shown. p-values are as indicated. Kaplan-Meier
analysis was per formed as we previously described in references 72 and 84. X-Tile software
was employed to identify subpopulation cut-points to observe maximum survival dierences
between the high expression and low expression subpopulations. The Log-rank test was used to
evaluate the signicance of dierences in survival curves for high vs. low signature-expressing
populations.
www.landesbioscience.com Cell Cycle 261
26. Rebbeck CA, Leroi AM, Burt A. Mitochondrial cap-
ture by a transm issible cancer. S cience 2011; 331:303;
PMID: 21252340; http://dx.doi.org /10.1126/ sci-
ence .119 7696 .
27. Spees JL, Olson SD, Whitney MJ, Prockop DJ.
Mitochondrial transfer between cells ca n rescue
aerobic respiration. Proc Natl Acad Sci USA 2006;
103:1283-8; PMID:16432190; http: //dx.doi.
org /10.1073/pna s.0510511103.
28. Berridge MV, Tan AS. Mitochondrial Gene Transfer
to Transplantable Tumors Lacking a Mitochondrial
Genome. Rejuvenation Res 2011; 14:13.
29. Martinez-Outschoorn UE, Pavlides S, W hita ker-
Menezes D, Daumer K M, Milliman JN, Chiavarina
B, et al. Tumor cells induce the cancer associ-
ated fibroblast phenotype via caveolin-1 degradation:
implications for breast cancer and DCIS therapy
with autophagy inhibitors. Cell Cycle 2010; 9:2423 -
33; PMID :20562526; http://dx.doi.org/10.4161/
cc.9.12.12048.
30. Martinez-Outschoorn UE, Lin Z, Trimmer C,
Flomenber g N, Wang C, Pavl ides S, et al. Ca ncer cells
metabol ically “ fertiliz e” the tumor mi croenvironme nt
with hydrogen perox ide, driving t he Warburg effect:
implications for PET imaging of huma n tumors. Cell
Cycle 2011; 10:2504-20; PMID:21778829; http://
dx.doi.or g/10.4161/cc.10.15.16585.
31. Lisanti MP, Martinez-Outschoorn UE, Lin Z,
Pavlides S, Whitaker-Menezes D, Pestell RG, et
al. Hydrogen peroxide fuels aging, infla mmation,
cancer metabolism and metastasis : the seed and soil
also needs “fertilizer”. Cell Cycle 2011; 10:2440-
9; PMID :21734470; http://dx.doi.org/10.4161/
cc.10.15.16870.
32. Lisanti MP, Martinez-Outschoorn UE, Pavlides S,
Whitaker-Menezes D, Pestell RG, Howell A, et
al. Accelerated aging in the tumor microenviron-
ment: connecting aging, inf lammation and cancer
metabolism with personalized medicine. Cell Cycle
2011; 10:2059-63; PMID :21654190; http ://d x.doi.
org /10.4161/cc.10.13.16233.
33. Martinez-Outschoorn UE, Balliet RM, Rivadeneira
DB, Chiavarina B, Pavlides S, Wang C, et al.
Oxid ative stress in c ancer asso ciated fibrobl asts drives
tumor-stroma co-evolution: A new pa radigm for
understanding tumor metabolism, the f ield effect
and genomic instability in cancer cells. Cell Cycle
2010; 9:3256-76; PMID:20814239; http ://d x.doi.
org /10.4161/cc.9.16.12553.
34. Martinez-Outschoorn UE, Trimmer C, Lin Z ,
Whitaker-Menezes D, Chiavarina B, Zhou J, et
al. Autophagy in cancer associated f ibroblasts pro-
motes tumor cell survival: Role of hypoxia, HIF1
induction and NFkappaB activation in the tumor
stromal microenvironment. Cell Cycle 2010; 9:3515-
33; PMID :20855962; http://dx.doi.org/10.4161/
cc.9.17.12928.
35. Martinez-Outschoorn UE , Whitaker-Menezes D,
Lin Z, Flomenberg N, Howell A, Pestell RG, et
al. Cytokine production and inf lammation drive
autophag y in the tumor microenvironment: role
of stromal caveolin-1 as a key regulator. Cell Cycle
2011; 10:1784-93; PMID:21566463 ; http://d x.doi.
org /10.4161/cc.10.11.15674.
36. Chiavarina B, Whitaker-Menezes D, Migneco G,
Martinez-Outschoorn UE , Pavlides S, Howell A,
et al. HIF1-alpha functions as a tumor promoter in
cancer associated f ibroblasts, and as a tumor suppres-
sor in breast cancer cells: Autophagy drives compart-
ment-specific oncogenesis. Cell Cycle 2010; 9:3534-
51; PMID:20864819; http://dx.doi.org/10.4161/
cc.9.17.12908.
37. Pavlides S, Whitaker-Menezes D, Ca stello-Cros R,
Flomenberg N, Witkiewicz AK, Frank PG, et al. The
reverse Warburg effect: aerobic glycolysis in cancer
associated f ibroblasts and the tumor stroma. Cell
Cycle 2009; 8: 3984- 4001; PMID:19923890; http://
dx.doi.org/10.4161/cc.8.23.10238.
Figure 12. Kaplan-Meier analysis of NRF1 target genes in breast cancer survival. Note that the
signature for NRF1 target genes predicts poor overall survival, especially in ER-positive patients.
Numbers of cases with annotation are shown. p-values are as indicated. X-Tile software was em-
ployed to identif y subpopulation cut-points to observe maximum survival dierences between
the high expression and low expression subpopulations. The Log-rank test was used to evaluate
the signicance of dierences in survival curves for high vs. low signature-expressing populations.
38. Pavlides S, Tsirigos A, Migneco G, Whitaker-
Menezes D, Chiavarina B, Flomenberg N, et al. The
autophagic tumor stroma model of cancer: Role of
oxidative stress and ketone production in fueling
tumor cell metabolism. Cell Cycle 2010; 9:3485-
505; PMID:20861672; http://dx.doi.org/10.4161/
cc.9.17.12721.
39. Pavlides S, Tsirigos A, Vera I, Flomenberg N, Frank
PG, Casimiro MC, et al. L oss of stromal caveolin-1
leads to oxidative stress, mimics hypoxia and drives
inflammation in the tumor microenvironment, con-
ferring the “reverse Warburg effect”: a transcrip-
tional i nformatics a nalysis wit h validation. C ell Cycle
2010; 9:2201-19; PMID:20519932; http://dx.doi.
org/10.4161/cc .9.11.118 48.
40. Pavlides S, Tsirigos A, Vera I, Flomenberg N, Frank
PG, Casimiro MC , et al. Transcriptional ev idence for
the “Reverse Warburg Effect” in hum an breast cancer
tumor stroma and metastasis : similarities with oxida-
tive stress, inflammation, Alzheimer’s disease and
“Neuron-Glia Metabolic Coupling”. Aging (Albany
NY) 2010; 2:185-99 ; PMID :20442453.
41. Martinez-Outschoorn UE, Prisco M, Ertel A,
Tsirigos A, Lin Z, Pavlides S, et al. Ketones and
lactate increa se cancer cell “stemness,” driving
recurrence, metastasis and poor clinical outcome
in breast cancer: achieving personalized medicine
via Metabolo-Genomics. Cell Cycle 2011; 10:1271-
86; PMID:21512313; http://dx.doi.org/10.4161/
cc.10.8.15330.
42. Martinez-Outschoorn UE, Lin Z, Ko Y H, Goldberg
AF, Flomenberg N, Wang C, et a l. Understanding
the metabolic basis of drug resistance: Therapeutic
induction of the Warburg effect kills cancer cells.
Cell Cycle 2011; 10:2521-8; PMID:21768775;
htt p: // dx .doi.org/10.4161/cc.10.15.1658 4.
43. Martinez-Outschoorn UE, Goldberg A, Lin Z, Ko
YH, Flomenberg N, Wang C, et al. A nti-estrogen
resistance in breast cancer is induced by the tumor
microenvironment and can be overcome by inhibit-
ing mitochondria l function in epithelial cancer cells.
Cance r Biol Ther 2011; 12:924 -38; PMID: 22041887;
htt p: // dx .doi.org/10.4161/cbt.12 .10.17780.
44. Bonuccelli G, Tsirigos A, Whitaker-Menezes D,
Pavlides S, Pestell RG, Chiavarina B, et al. Ketones
and lactate “fuel” tumor growth and metastasis:
Evidence that epithelial cancer cells use oxidative
mitochondrial metabolism. Cell Cycle 2010; 9 :3506-
14; PMID:20818174; http://dx.doi.org/10.4161/
cc.9.17.12731.
45. Bonuccelli G, Whitaker-Menezes D, Castello-Cros
R, Pavlides S, Pestell RG, Fatatis A, et al. The reverse
Warburg effect: glycolysis inhibitors prevent the
tumor promoting effects of caveolin-1 deficient can-
cer associated fibroblasts. Cell Cycle 2010; 9:1960-
71; PMID :20495363 ; http://dx.doi.org/10.4161/
cc.9.10.11601.
46. Ni Chonghaile T, Sarosiek KA, Vo TT, Ryan JA,
Tammareddi A, Moore Vdel G, et al. Pretreatment
mitochondrial priming correlates with clinical
response to cytotoxic chemotherapy. Science 2011;
334:1129-33; PMID :22033517; http://dx.doi.
org /10.112 6/ science.1206727.
47. El-Mir MY, Nogueira V, Fontaine E, Averet N,
Rigou let M, Leverve X. Dimethylbiguanide inhib-
its cell respiration via an indirect effect targeted
on the respiratory chain complex I. J Biol Chem
2000 ; 275:223-8; PMID:10617608; http://dx.doi.
org /10.1074/jbc.275.1.2 23.
262 Cell Cycle Volume 11 Issue 2
56. Xu D, Finkel T. A role for mitochondria as potential
regulators of cellular life span. Biochem Biophys
Res Commun 2002 ; 294:245-8 ; PMID :12051701;
http://dx.doi.org/10.1016/S0006-291X(02)00464-3.
57. Bulterijs S. Met formin a s a geroprot ector. Rejuve nation
Res 2011; 14:469-82; PMID:21882902; http://
dx .doi.or g/10 .10 89 /re j. 2011.1153.
58. Anisimov VN, Berstein LM, Popovich IG,
Zabezhinski MA, Egormin PA, Piskunova TS, et
al. If started early in life, metformin treatment
increases life span and postpones tumors in female
SHR mice. Aging (Albany NY ) 2011; 3:148-57;
PMID :21386129.
59. A nisimov VN, Piskunova TS, Popovich IG,
Zabezhinski MA, Tyndyk ML, Egormin PA, et al.
Gender differences in metformin effect on aging,
life span and spontaneous tumorigenesis in 129/
Sv mice. Aging (A lbany N Y) 2010; 2:945-58;
PMID : 2116 4 223.
60. Anisimov V N. Metformin for aging and cancer
prevention. Aging (Albany NY) 2010; 2 :760-74;
PMID: 2108 4729.
61. Mouchiroud L , Molin L , Dalliere N, Solari F. Life
span extension by resveratrol, rapamycin and met-
formin: The promise of dietary restriction mimet-
ics for an healthy a ging. Biofactors 2010; 36:377-
82; PMID:20848587; http://dx.doi.org/10.1002/
biof.127.
62. Anisimov VN, Egormin PA, Piskunova TS, Popovich
IG, Tyndyk ML, Yurova MN, et al. Metformin
extends life span of HER-2/neu transgenic mice
and in combination with melatonin inhibits
growth of transplantable tumors in vivo. Cell Cycle
2010; 9:188-97; PMID:20016287; http://dx.doi.
org /10.4161/cc.9.1.10407.
63. Anisimov V N, Berstein LM, Egormin PA, Piskunova
TS, Popovich IG, Zabezhinski MA, et al. Metformin
slows dow n aging and ex tends life spa n of female SHR
mice. Cell Cycle 2008; 7:2769-73; PMID :18728386 ;
ht t p : // d x . doi . o rg /10.4 16 1/c c . 7.1 7.662 5.
64. Anisimov VN, Egormin PA, Bershtein L M,
Zabezhinskii M A, Piskunova TS, Popovich IG,
et al. Metformin decelerates aging and develop-
ment of mammary tumors in HER-2/neu trans-
genic mice. Bull Exp Biol Med 2005; 139:721-3;
PMID:16224592 ; ht tp: //d x.doi.or g/10.1007/s10517-
005-0389-9.
65. Anisimov V N, Berstein LM, Egormin PA, Piskunova
TS, Popovich IG, Zabezhinski MA, et al. Effect
of metformin on life span and on the develop-
ment of spontaneous ma mmary tumors in HER-2/
neu transgenic mice. Exp Gerontol 2005; 40: 685-
93; PMID :16125352; http://dx.doi.org/10.1016/j.
exger.2005.07.007.
66. Inoue S, Hasegawa K, Ito S, Wakamatsu K, Fujita K .
Antimelanoma activity of chloroquine, an antima-
laria l agent with high affinity for melanin. Pigment
Cell Res 1993; 6:354-8 ; PMID :8302774; http://
dx .doi.or g/10 .1111/ j.1600 - 0749.1993.tb 00613. x.
67. Paget S. The distribution of secondary growths in
cancer of the breast. La ncet 1889; 133:571-3; http://
dx.doi.org/10.1016/S0140-6736(00)49915-0.
68. Hart IR. ‘Seed a nd soil’ revisited: mechanisms of
site-specific metastasis. Cancer Metastasis Rev 1982;
1:5-16; PMID :6764375; http://dx.doi.org/10.1007/
BF00049477.
69. Hart IR, Fidler IJ. Role of organ selectivity in the
determination of metastatic patterns of B16 melano-
ma. Cancer Res 1980; 40 :2281-7; PMID:7388794.
70. Hart IR, Talmadge JE, Fidler IJ. Metastatic behav-
ior of a murine reticulum cell sarcoma exhibiting
organ-specif ic growth. Cancer Res 1981; 41:1281-7;
PMID :7011533.
71. Nieman KM, Kenny HA, Penicka CV, Ladanyi A,
Buell-Gutbrod R, Zillhardt MR, et al. Adipocytes
promote ovarian cancer metastasis and provide ener-
gy for rapid tumor growth. Nat Med 2011; 17:1498-
503; PMID:22037646; http://dx.doi.org/10.1038/
nm.2492.
52. Zakikhani M, Dowling RJ, Sonenberg N, Pollak
MN. The effects of adiponectin and metformin
on prostate and colon neoplasia involve activation
of AMP-activated protein kinase. Cancer Prev Res
(Phila) 2008 ; 1:369-75; PMID:19138981; http://
dx.doi.org/10.1158/1940-6207.CAPR-08-0081.
53. Reliene R, Schiestl R H. Antioxidant N-acetyl c ys-
teine reduces incidence and multiplicit y of lym-
phoma in Atm deficient mice. DNA Repair (Amst)
2006 ; 5: 852-9; PMID:16781197; http://dx.doi.
org /10.1016 /j.dnarep.2006. 05.003.
54. Flurkey K, Ast le CM, Harrison DE. Life extension by
diet res triction and N-acetyl-L-c ysteine in gene tically
heterogeneous mice. J Gerontol A Biol Sci Med Sci
2010; 65:1275-84; PMID:20819793; http ://d x.doi.
org /10.1093/gerona/ glq155.
55. Brack C, Bechter-Thuring E, Labuhn M.
N-acet ylcysteine slows down aging and increases the
life span of Drosophila melanogaster. Cell Mol Life
Sci 1997; 53:960-6 ; PMID:9447249; http://dx.doi.
org/10.1007/PL00013199.
48. Libby G, Donnelly L A, Donnan PT, Alessi DR,
Morris AD, Evans JM. New users of metformin
are at low risk of incident cancer: a cohort study
among people with type 2 diabetes. Diabetes Care
2009; 32:1620-5; PMID:19564453; http://dx.doi.
org /10.2337/dc08 -2175.
49. Jira lerspong S, Palla SL, Giordano SH, Meric-
Bernst am F, Liedtke C, B arnett CM, et a l. Metformin
and pathologic complete responses to neoadjuvant
chemotherapy in diabetic patients with breast cancer.
J Clin Oncol 2009; 27:3297-302; PMID:19487376;
htt p: // dx .doi.org/10.1200/JCO.20 09.19.6410.
50. Phoenix KN, Vumbaca F, Fox MM, Evans R,
Claffey KP. Dietary energy availability a ffects pri-
mary and meta static breast cancer and metformin
efficacy. Breast Cancer Res Treat 2010; 123:333-
44; PMID:20204498; http://dx.doi.org/10.1007/
s10549-009-0647-z.
51. Riccio A, Del Prato S, Vigili de Kreutzenberg S,
Tiengo A. Glucose and lipid metabolism in non-
insulin-dependent diabetes. Effect of metformin.
Diabete Metab 1991; 17:180-4; PMID:1936473.
Figure 13. Kaplan-Meier analysis of NRF1 target genes in ER+/Luminal A breast cancers. Note that
the signature for NRF1 target genes predicts metastasis, recurrence, and poor overall survival,
in ER-positive/Luminal A breast cancer patients. Numbers of cases with annotation are shown.
p-values are as indicated. X-Tile sof tware was employed to identif y subpopulation cut-points
to observe maximum survival dierences between the high expression and low expression
subpopulations. The Log-rank test was used to evaluate the signicance of dierences in survival
curves for high vs. low signature-expressing populations.
www.landesbioscience.com Cell Cycle 263
72. W hita ker-Menezes D, Martinez-Outschoorn UE,
Flomenberg N, Birbe RC, Witkiewicz A K, Howell
A, et al. Hyperactivation of Oxidative Mitochondrial
Metabolism in Epithelial Cancer Cells In Situ:
Visua lizing the Therapeutic Effects of Metformin
in Tumor Tissue. Cell Cycle 2011; 10:4047-64;
PMI D:22134189.
73. Sahin E, Colla S, Liesa M, Moslehi J, Mul ler FL, Guo
M, et al. Telomere dy sfunction i nduces metaboli c and
mitochondrial compromise. Nature 2011; 470:359-
65; PMID: 21307849; http://dx.doi.org /10.1038/
nature09787.
74. David R. aging: Mitochondria and telomeres come
together. Nat Rev Mol Cell Biol 2011; 12:204;
PMID: 21407239; http: //dx .doi.org/10.1038/
nrm3082.
75. Kelly DP. Cell biology: aging theories unif ied.
Nature 2011; 470:342-3 ; PMID:21307852; http://
dx.doi.or g/10.1038 /n ature09896.
76. Finkel T. Telomeres and mitochondrial function.
Circ Res 2011; 108:903-4; PMID:21493920; http://
dx.doi.org/10.1161/RES.0b 013e31821bc2d8.
77. Buzzai M, Jones RG, Amaravadi RK, Lum JJ,
DeBerardinis RJ, Zhao F, et al. Systemic treatment
with the antidiabetic drug metformin selectively
impairs p53-def icient tumor cell growt h. Cancer Res
2007; 67:6745-52; PMID :17638885; http://dx.doi.
org/10.1158/0008-5472.CAN-06-4447.
78. Bhalla K, Hwang BJ, Dewi R E, Ou L, Twaddel W,
Fang HB, e t al. PGC1alpha Promote s Tu mor Growth
by Inducing Gene Expression Programs Supporting
Lipogenesis. Ca ncer Res 2011; 71:6888-98;
PMID: 21914785; http ://d x.doi.org /10.1158/0008-
5472.C A N-11-1011.
79. Shiot a M, Yokomizo A, Tada Y, Inoku chi J, Tatsugam i
K, Kuroi wa K, et al. Peroxis ome proliferator-act ivated
receptor gamma coactivator-1alpha interacts with the
androg en receptor (AR ) and promotes prostate canc er
cell growth by activating the AR. Mol Endocrinol
2010; 24:114-27; PMID:19884383; http://dx.doi.
org /10.1210/me.2009-03 02.
80. Cormio A, Guerra F, Cormio G, Pesce V, Fracasso F,
Loizzi V, et al. The PGC -1alpha-dependent pathway
of mitochondrial biogenesis is upregulated in type I
endometrial ca ncer. Biochem Biophys Res Commun
2009; 390:1182-5; PMID:19861117; http://dx.doi.
org/10.1016/j.bbrc.2009.10.114.
81. Škrtic M, Sriskanthadevan S, Jhas B, Gebbia M,
Wang X, Wang Z, et al. Inhibition of mitochon-
drial translation a s a therapeutic strateg y for human
acute myeloid leukemia. Cancer Cell 2011; 20: 674-
88; PMID:22094260 ; http://d x.doi.org/10.1016/j.
ccr.2011.10.015.
82. Järås M, Ebert BL. Power cut: inhibiting mitochon-
drial translation to target leukemia. Cancer Cell
2011; 20:555-6 ; PMID:22094249; http: //dx.doi.
org /10.1016 /j.ccr.2011.10.028.
83. Casey T, Bond J, Tighe S, Hunter T, Lintault L, Patel
O, et al. Molecular signatures suggest a major role
for stroma l cells in development of invasive breast
cancer. Breast Cancer Res Treat 2009; 114:47-62 ;
PMID:18373191; htt p: //dx .doi.org/10.1007/s10549-
008-9982-8.
84. Ertel A, Dean JL, Rui H, Liu C, Witkiewicz AK,
Knudsen KE, et al. R B-pathway disruption in breast
cancer: dif ferential association with disease subtypes,
disease-specific prognosis and therapeutic response.
Cell Cycle 2010; 9: 4153-63; PMID:20948315;
htt p: // dx .doi.org/10.4161/cc.9.20.13 454 .
85. W hita ker-Menezes D, Martinez-Outschoorn UE,
Lin Z, Ertel A, Flomenberg N, Witkiewicz AK, et
al. Evidence for a stromal-epithelial “lactate shuttle
in human tumors : MCT4 is a marker of oxidative
stress in cancer-associated fibroblasts. Cell Cycle
2011; 10:1772-83; PMID:21558814; http: //dx.doi.
org /10.4161/cc.10.11.15659.
Figure 14. NRF1, a positive regulator of mitochondrial biogenesis, is preferentially expressed in
human epithelial cancer cells in breast cancer patients. Paran-embedded sections of human
breast cancer samples were immunostained with antibodies directed against NRF1 (sc-33772;
Santa Cruz Biotech, Inc.). Slides were then counter-stained with hematoxylin (blue color). Note
that NRF1 (brown color) is highly expressed in the epithelial compartment of human breast
cancers. Two representative images are shown. Original magnication, 40x and 60x, as indicated.
Immunostaining was performed essentially as previously described in references 72 and 85.
Figure 15. NRF1 is highly expressed in epithelial cancer cell nests, in breast cancer patients. Fresh
frozen sections of human breast cancer samples were immunostained with antibodies directed
against NRF1 (sc-33772; Santa Cruz Biotech, Inc.). Slides were then counter-stained with hema-
toxylin (blue color). NRF1 (brown color) is highly expressed in the epithelial compartment of
human breast cancers, relative to adjacent stromal cells. Three representative images are shown.
Original magnication, 20x, 40x and 60x, as indicated. Immunostaining was performed essentially
as previously described in references 72 and 85.
    • "Using an shRNA approach we proved that the overexpression of these master transcription factors are crucial in the AICAR+MTX-mediated inhibition of proliferation and the upregulation of PGCs and FOXO1 upon AMPK activation explains the upregulation of mitochondrial activity. Importantly, an in silico approach has already nominated PGCs and NRF1 as key proteins regulating bioenergetics in breast cancer cells [43]. Furthermore, Faubert and colleagues [10] have shown the suppression of the HIF and mTORC1 pathway in experimental lymphoma models upon AICAR treatment that may be implicated in our models too. "
    [Show abstract] [Hide abstract] ABSTRACT: Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK) jointly with methotrexate (MTX), a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1β or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer.
    Full-text · Article · Feb 2016
    • "Similarly, we have previously shown that markers of mitochondrial mass and mitochondrial activity are specifically localized to the basal stem cell layer in normal human mucosa, which co-localizes with Ki67, an established marker of cell proliferation [40]. In addition, this mitochondria-rich population of cells is dramatically expanded in head and neck cancers [40] and breast cancers414243. Moreover, recombinant over-expression of mitochondrial-related proteins, such as PGC1-alpha/ beta, POLRMT, MitoNEET or GOLPH3, is sufficient to promote tumor growth, by up to 3-fold, in xenografted pre-clinical models of human breast cancers [44, 45]. "
    [Show abstract] [Hide abstract] ABSTRACT: Here, we developed an isogenic cell model of “stemness” to facilitate protein biomarker discovery in breast cancer. For this purpose, we used knowledge gained previously from the study of the mouse mammary tumor virus (MMTV). MMTV initiates mammary tumorigenesis in mice by promoter insertion adjacent to two main integration sites, namely Int-1 (Wnt1) and Int-2 (Fgf3), which ultimately activates Wnt/β-catenin signaling, driving the propagation of mammary cancer stem cells (CSCs). Thus, to develop a humanized model of MMTV signaling, we over-expressed WNT1 and FGF3 in MCF7 cells, an ER(+) human breast cancer cell line. We then validated that MCF7 cells over-expressing both WNT1 and FGF3 show a 3.5-fold increase in mammosphere formation, and that conditioned media from these cells is also sufficient to promote stem cell activity in untransfected parental MCF7 and T47D cells, as WNT1 and FGF3 are secreted factors. Proteomic analysis of this model system revealed the induction of i) EMT markers, ii) mitochondrial proteins, iii) glycolytic enzymes and iv) protein synthesis machinery, consistent with an anabolic CSC phenotype. MitoTracker staining validated the expected WNT1/FGF3-induced increase in mitochondrial mass and activity, which presumably reflects increased mitochondrial biogenesis. Importantly, many of the proteins that were up-regulated by WNT/FGF-signaling in MCF7 cells, were also transcriptionally over-expressed in human breast cancer cells in vivo, based on the bioinformatic analysis of public gene expression datasets of laser-captured patient samples. As such, this isogenic cell model should accelerate the discovery of new biomarkers to predict clinical outcome in breast cancer, facilitating the development of personalized medicine. Finally, we used mitochondrial mass as a surrogate marker for increased mitochondrial biogenesis in untransfected MCF7 cells. As predicted, metabolic fractionation of parental MCF7 cells, via MitoTracker staining, indicated that high mitochondrial mass is a new metabolic biomarker for the enrichment of anabolic CSCs, as functionally assessed by mammosphere-forming activity. This observation has broad implications for understanding the role of mitochondrial biogenesis in the propagation of stem-like cancer cells. Technically, this general metabolic approach could be applied to any cancer type, to identify and target the mitochondrial-rich CSC population. The implications of our work for understanding the role of mitochondrial metabolism in viral oncogenesis driven by random promoter insertions are also discussed, in the context of MMTV and ALV infections.
    Full-text · Article · Sep 2015
    • "glutaminolysis) in many cancers [21, 22]. Cancer cells often have increased oxidative phosphorylation (OXPHOS) and elevated uptake and consumption of glutamine [23, 24]. Many cancer cells become addicted to glutamine since it is readily available in high amounts in the circulation and is actively taken up by the cells [25, 26]. "
    [Show abstract] [Hide abstract] ABSTRACT: Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets.
    Full-text · Article · Sep 2015
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