Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis
A single cell has the potential to kill an entire human being. Efforts to cure cancer are limited by survival of individual cancer cells despite immune surveillance and toxic therapies. Understanding the intricate network of pathways that maintain cellular homeostasis and mediate stress response or...
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| Cite this: | Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis / D.M. Benbrook, A. Long // Experimental Oncology. — 2012. — Т. 34, № 3. — С. 286-297. — Бібліогр.: 129 назв. — англ. |
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Benbrook, D.M. Long, A. 2018-06-19T18:49:02Z 2018-06-19T18:49:02Z 2012 Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis / D.M. Benbrook, A. Long // Experimental Oncology. — 2012. — Т. 34, № 3. — С. 286-297. — Бібліогр.: 129 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/139052 A single cell has the potential to kill an entire human being. Efforts to cure cancer are limited by survival of individual cancer cells despite immune surveillance and toxic therapies. Understanding the intricate network of pathways that maintain cellular homeostasis and mediate stress response or default into cell death is critical to the development of strategies to eradicate cancer. Autophagy, proteasomal degradation and the unfolded protein response (UPR) are cellular pathways that degrade and recycle excess or damaged proteins to maintain cellular homeostasis and survival. This review will discuss autophagy and how it is integrated with proteasomal degradation and UPR to govern cell fate through restoration of cellular homeostasis or default into the apoptotic cell death pathway. The first response of autophagy is macroautophagy, which sequesters cytoplasm including organelles inside double-membraned autophagosome vesicles that fuse with lysosomes to degrade and recycle the contents. Ubiquitination patterns on proteins targeted for degradation determine whether adapter proteins will bring them to developing autophagosomes or to proteasomes. Macroautophagy is followed by chaperone-mediated autophagy (CMA), in which Hsc70 (Heat shock cognate 70) selectively binds proteins with exposed KFERQ motifs and pushes them inside lysosomes through the LAMP-2A (Lysosome-associated membrane protein type 2A) receptor. These two processes and the lesser understood microautophagy, which involves direct engulfment of proteins into lysosomes, occur at basal and induced levels. Insufficient proteasome function or ER stress induction of UPR can induce autophagy, which can mitigate damage and stress. If this network is incapable of repairing the damage or overcoming continued stress, the default pathway of apoptosis is engaged to destroy the cell. Induction of macroautophagy by cancer therapeutics has led to clinical trials investigating combinations of HCQ (hydroxychloriquine) suppression of autophagy with apoptosis-inducing agents. Further study of the complex integration of autophagy, proteasomal degradation, UPR and apoptosis is likely to provide additional targets for our fight against cancer. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Reviews Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis Article published earlier |
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Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis |
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Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis Benbrook, D.M. Long, A. Reviews |
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Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis |
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Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis |
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Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis |
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integration of autography, proteasomal degradation, unfolded protein responce and apoptosis |
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Experimental Oncology |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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A single cell has the potential to kill an entire human being. Efforts to cure cancer are limited by survival of individual cancer cells despite immune surveillance and toxic therapies. Understanding the intricate network of pathways that maintain cellular homeostasis and mediate stress response or default into cell death is critical to the development of strategies to eradicate cancer. Autophagy, proteasomal degradation and the unfolded protein response (UPR) are cellular pathways that degrade and recycle excess or damaged proteins to maintain cellular homeostasis and survival. This review will discuss autophagy and how it is integrated with proteasomal degradation and UPR to govern cell fate through restoration of cellular homeostasis or default into the apoptotic cell death pathway. The first response of autophagy is macroautophagy, which sequesters cytoplasm including organelles inside double-membraned autophagosome vesicles that fuse with lysosomes to degrade and recycle the contents. Ubiquitination patterns on proteins targeted for degradation determine whether adapter proteins will bring them to developing autophagosomes or to proteasomes. Macroautophagy is followed by chaperone-mediated autophagy (CMA), in which Hsc70 (Heat shock cognate 70) selectively binds proteins with exposed KFERQ motifs and pushes them inside lysosomes through the LAMP-2A (Lysosome-associated membrane protein type 2A) receptor. These two processes and the lesser understood microautophagy, which involves direct engulfment of proteins into lysosomes, occur at basal and induced levels. Insufficient proteasome function or ER stress induction of UPR can induce autophagy, which can mitigate damage and stress. If this network is incapable of repairing the damage or overcoming continued stress, the default pathway of apoptosis is engaged to destroy the cell. Induction of macroautophagy by cancer therapeutics has led to clinical trials investigating combinations of HCQ (hydroxychloriquine) suppression of autophagy with apoptosis-inducing agents. Further study of the complex integration of autophagy, proteasomal degradation, UPR and apoptosis is likely to provide additional targets for our fight against cancer. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”.
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1812-9269 |
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https://nasplib.isofts.kiev.ua/handle/123456789/139052 |
| citation_txt |
Integration of autography, proteasomal degradation, unfolded protein responce and apoptosis / D.M. Benbrook, A. Long // Experimental Oncology. — 2012. — Т. 34, № 3. — С. 286-297. — Бібліогр.: 129 назв. — англ. |
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286 Experimental Oncology 34, 286–297, 2012 (September)
INTEGRATION OF AUTOPHAGY, PROTEASOMAL DEGRADATION,
UNFOLDED PROTEIN RESPONSE AND APOPTOSIS
D.M. Benbrook1,2,*, A. Long2
1Department of Obstetrics and Gynecology and 2Department of Biochemistry and Molecular Biology
University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
A single cell has the potential to kill an entire human being. Efforts to cure cancer are limited by survival of individual cancer cells
despite immune surveillance and toxic therapies. Understanding the intricate network of pathways that maintain cellular homeo-
stasis and mediate stress response or default into cell death is critical to the development of strategies to eradicate cancer. Au-
tophagy, proteasomal degradation and the unfolded protein response (UPR) are cellular pathways that degrade and recycle excess
or damaged proteins to maintain cellular homeostasis and survival. This review will discuss autophagy and how it is integrated with
proteasomal degradation and UPR to govern cell fate through restoration of cellular homeostasis or default into the apoptotic cell
death pathway. The first response of autophagy is macroautophagy, which sequesters cytoplasm including organelles inside double-
membraned autophagosome vesicles that fuse with lysosomes to degrade and recycle the contents. Ubiquitination patterns on pro-
teins targeted for degradation determine whether adapter proteins will bring them to developing autophagosomes or to proteasomes.
Macroautophagy is followed by chaperone-mediated autophagy (CMA), in which Hsc70 (Heat shock cognate 70) selectively binds
proteins with exposed KFERQ motifs and pushes them inside lysosomes through the LAMP-2A (Lysosome-associated membrane
protein type 2A) receptor. These two processes and the lesser understood microautophagy, which involves direct engulfment of pro-
teins into lysosomes, occur at basal and induced levels. Insufficient proteasome function or ER stress induction of UPR can induce
autophagy, which can mitigate damage and stress. If this network is incapable of repairing the damage or overcoming continued
stress, the default pathway of apoptosis is engaged to destroy the cell. Induction of macroautophagy by cancer therapeutics has led
to clinical trials investigating combinations of HCQ (hydroxychloriquine) suppression of autophagy with apoptosis-inducing agents.
Further study of the complex integration of autophagy, proteasomal degradation, UPR and apoptosis is likely to provide addi-
tional targets for our fight against cancer. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”.
Key Words: autophagy, proteasomal degradation, endoplasmic reticulum stress, unfolded protein response, apoptosis.
INTRODUCTION
Understanding how cells die is critical knowledge
needed for the development of health care strategies
to prevent the death of cells in degenerative and acute
diseases and to induce the death of diseased cells that
can harm the body if not eliminated. Both morphologi-
cal and molecular events that occur in dying cells have
been characterized and categorized into 13 current
modes [37, 68]. More recently, an iron-dependent
form of non-apoptotic death termed “ferroptosis” has
been described [30]. Autophagy, which was officially
identified and named by Christian de Duve in 1963 [22,
59], is formally classified as a form of cell death, how-
ever the majority of evidence implicates autophagy
as a mechanism to maintain cellular homeostasis and
recover from stress, while unequivocal evidence that
autophagy causes cell death is rare. The survival func-
tion is accomplished through digestion of long-lived
or damaged proteins and organelles and release of the
components for recycling. The death function is be-
lieved to be caused by excessive digestion of cellular
components or selective digestion of survival factors
over death factors. This review will discuss the integra-
tion of autophagy with other cellular processes that
maintain homeostasis and mediate stress responses
leading to cancer cell survival or death, sometimes
through apoptosis, and how these mechanisms are
being targeted to improve cancer therapy. For more
details on the mechanism of autophagy, readers are
directed to several recent reviews [7, 75, 76, 112, 123].
FORMS OF AUTOPHAGY
There are three natural processes of autophagy
in the cell, MA (macroautophagy), CMA (chaperone-
mediated autophagy) and microautophagy. Mac-
roautophagy is a natural process in which portions
Received: June 15, 2012
*Correspondence: Fax: (405) 271-3874
E-mail: Doris-Benbrook@ouhsc.edu
Abbreviations used: AMP — adenosine 5’-monophosphate; AMPK —
AMP kinase; ATF — activating transcription factor; ATG — autophagy-
related genes; BAG-1 — Bcl-2-associated athanogene 1; Bcl-2 —
B-Cell CLL/Lymphoma 2; CHOP — CCAAT/-enhancer- binding protein
homologous protein; CMA — chaperone-mediated autophagy; CQ —
chloroquine; EF1α — elongation factor 1α; ER — endoplasmic reticulum;
ERAD — ER-associated degradation; FIP200 — focal adhesion kinase
interacting protein of 200 kD; GADD — growth arrest and DNA damage;
Gal3 — galectin-3; GFAP — glial fibrillary acidic protein; GTPases — gua-
nosine triphosphatases; HCQ — hydroxychloroquine; HDAC6 — histone
deacetylase 6; HLA — human leukocyte antigen; HMGB1 — high mobil-
ity group box 1; IGF1 — insulin like growth factor 1; IRE1α — inositol-
requiring enzyme 1α; LAMP-2A — lysosome-associated membrane
protein type 2A; LC3 — light chain of the microtubule-associated protein
1; MDM2 — murine double minute 2; MEFs — murine embryonic fibro-
blasts; MLIV — mucolipidosis Type IV; mTOR — mammalian target of ra-
pamycin; NBR1 — neighbor of BRC1; PE — phosphatidylethanoamine;
PERK — double-stranded RNA-dependent protein kinase (PKR)-like
ER kinase; PI3K — phosphoinositol 3-kinase; PIP3 — PtdIns(3,4,5)P3;
PMN — piecemeal microautophagy of the nucleus; PS — phosphoati-
dylserine; Rheb — Ras homolog enriched in brain; TRPML1 — transient
receptor potential mucolipin-1; TSC — tuberous sclerosis complex;
ULK — uncoordinated 51-like kinase; UPR — unfolded protein response;
V-ATPase — vacuolar H+ ATPase; Vps — vacuolar protein sorting.
Exp Oncol 2012
34, 3, 286–297
INVITED REVIEW
Experimental Oncology 34, 286–297, 2012 (September)34, 286–297, 2012 (September) (September) 287
of the cytoplasm, including long-lived proteins and
organelles, are sequestered inside double-membraned
vesicles called autophagosomes. The autophagosomes
eventually fuse with lysosomes to form autophagoly-
sosomes where the contents are digested and their
components released for recycling within the cells. CMA
is driven by Hsc70 (heat shock cognate 70, also called
HSPA8 [heat shock protein A8]), which binds specific
proteins and transports them directly into the lysosome
[76]. In microautophagy, defective molecules or organ-
elles are directly engulfed into the lysosomes for degra-
dation and recycling of their components [75]. All three
forms of autophagy occur at basal levels to maintain
the cell, while suprabasal levels are induced by nutrient
or oxygen deprivation, endoplasmic reticulum stress,
proteasome malfunction or damage caused by drugs
or radiation. Most articles refer to macroautophagy
as autophagy, however because this article addresses
all three forms of autophagy in detail, the term macro-
autophagy is used herein to describe the specific pro-
cess of autophagosome-mediated recycling. The term
autophagy is used in situations where it is feasible that
all three forms of autophagy could be actively involved
in the process under discussion.
MECHANISM, MEASUREMENT AND
MANIPULATION OF MACROAUTOPHAGY
The production and processing of autophagic ves-
icles is divided into 4 steps: 1) initiation, 2) nucleation,
3) maturation and 4) fusion with lysosomes (Fig. 1).
These processes are mediated by a series of proteins
encoded by autophagy-related genes (ATGs), which
were originally characterized in yeast, and are highly
conserved in higher eukaryotes [60].
During initiation, de novo synthesis of isolation
membranes recruits lipids from several organelles
depending on the cell type and stimulus. Electron
micrograph documentation of ER located on both
sides of isolation membranes, and what appears
to be a single bridge connecting the ER to the isola-
tion membrane, indicate that this organelle is a source
of membrane lipids for the de novo formation
of autophagosomes in the cytoplasm [42]. Formation
of isolation membranes from the mitochondria ap-
pears to be dependent upon PS (phosphatidylserine)
transfer from the ER to the mitochondria [41]. The
mitochondrial enzyme, PS decarboxylase, converts
PS to PE (phosphatidylethanolamine), which is needed
for autophagosome formation as described below
[113]. Proteins associated with only the outer leaflet
of the mitochondrial membrane appear to transfer
to the autophagosome, while proteins that traverse the
entire mitochondrial membrane are retained in the mi-
tochondria [41]. The plasma membrane has also been
shown to contribute lipids to the initiation membrane
through a process dependent upon the interaction
of clatherin on the plasma membrane with Atg16L
on the forming autophagosome [100]. An alternative
form of macroautophagy shown to occur during fetal
development and erythroid maturation derives mem-
brane lipids for de novo autophagosome formation
from the trans-Golgi and late endosomes [87].
The complex of proteins that mediate initiation con-
sists of ULK1 (uncoordinated 51-like kinase 1/Atg1),
Atg13 and FIP200 (focal adhesion kinase interacting
protein of 200 kD/Atg17). Basal levels of macroau-
tophagy are kept in check by mTORC1 (mamma-
lian target of rapamycin complex 1) phosphorylation
of Atg13 and ULK1, which inhibits ULK1 phosphoryla-
tion of FIP200 [12, 38, 44, 49]. The mTORC1 complex
is an important component of a network that senses
the nutrient state of the cell and accordingly controls
the levels of anabolism and catabolism to maintain
homeostasis [46] (Fig. 2). High levels of amino acids
maintain mTORC1 in an active state by enhancing bind-
ing of this complex to regulatory proteins Rag and Rheb
(Ras homolog enriched in brain) GTPases (guanosine
triphosphatases) [56, 105]. Insulin and IGF1 (insulin
like growth factor 1) indirectly induce mTORC1 activity
by stimulating class 1 PI3K (phosphoinositol 3-kinase)
production of PIP3 (PtdIns(3,4,5)P3), which induces
the Akt kinase at the plasma membrane, which in turn
activates mTORC1 by inhibiting TSC (tuberous sclero-
sis complex) proteins 1 and 2, thereby relieving their
repression of Rheb [4, 8]. Low glucose levels or high
levels of AMP (adenosine 5’-monophosphate), which
indicate low cellular energy status or stress, activate
AMPK (AMP-activated protein kinase), which inhibits
mTORC1 and stimulates macroautophagy [46, 95].
In summary, high levels of amino acids, insulin and
IGF-1 inhibit macroautophagy by inducing the PI3K/
Akt/mTORC1 pathway, while low glucose and high
AMP levels induce autophagy by activating the AMPK
and repressing mTORC1 activity (Fig. 2). Chemical
inhibitors of mTORC1 currently in clinical use or in clini-
cal trials, including rapamycin and analogs called
Fig. 1. Simplified Illustration of macroautophagy
288 Experimental Oncology 34, 286–297, 2012 (September)
rapalogs, such as Everolimus (RAD001), induce
macroautophagy and are often used as tools to study
autophagy [10, 57]. As discussed below, this induction
of macroautophagy interferes with the clinical efficacy
of these drugs as anti-cancer agents.
Nucleation is controlled by a class III PI3K called
Vps34 (vacuolar protein sorting 34) that forms a com-
plex with Beclin 1 (Atg6/Vp330), p150 (Vps15) and
Atg14L [79]. Production of PIP3 by Vps34 recruits WIPI
(WD40 repeat protein Interacting with phosphoInositi-
des/Atg18) proteins to the isolation membrane allow-
ing recruitment of LC3 (light chain of the microtubule-
associated protein 1/ Atg8) and further evolution of the
autophagosome [97]. While specific inhibition of class
I PI3K stimulates macroautophagy indirectly through
downstream Akt/mTOR inhibition, specific inhibi-
tion of class III PI3K Vps34 inhibits macroautophagy
through reduction of an autophagy-specific PIP3 pool
[95]. As discussed below, nucleation can also be inhib-
ited by binding of the anti-apoptotic protein Bcl-2 (B-
Cell CLL/Lymphoma 2) to Beclin 1 [94].
Two interdependent ubiquitin-like conjugation sys-
tems mediate the maturation (elongation, curvature
and closure) of the autophagosome. In one system,
LC3 is first cleaved by the Atg4 serine protease and
then conjugated to PE by the Atg7 and Atg3 enzymes
[45]. The unmodified and lipidated forms of LC3 are
termed LC3-I and LC3-II, respectively, and can be dis-
tinguished by Western blot analysis, a well accepted
method to monitor macroautophagy [6]. The second
system recruits LC3-II to the isolation membrane by the
ubiquitin-like activity of Atg12, which is covalently bound
to Atg5 and physically associated with Atg-16L to form
a complex [86]. Once the isolation membrane is formed,
the Atg-12, -5, -16L complex is released, which affords
it’s utilization as a marker of isolation membrane forma-
tion [86]. LC3-II on the other hand, remains associated
with the autophagosome until fusion with the lyso-
some, and the transition of diffuse to punctuate pattern
of a transfected LC3-green fluorescent protein fusion
protein in the cytoplasm is a commonly used marker
of autophagosome formation [6, 86].
Before fusion with the lysosome, ubiquitinated
proteins can be brought to the inside of the develop-
ing autophagosome by adapter proteins. Proteins are
targeted for degradation by E1, E2 and E3 ubiquiti-
nases that attach the C-terminal glycine on ubiquitin
to the ε-amino group of a lysine residue in the protein
being targeted [96]. Multiple molecules of ubiquitin
can be added to the same protein as individual com-
ponents, and to Lys residues on existing ubiquitins
that are already present on the target proteins to form
branched chains [119]. Adapter proteins/macro-
autophagy receptors called p62, NBR1 (neighbor
of BRC1), HDAC6 (histone deacetylase 6) and Alfy bind
the ubiquitinated proteins and bring them inside devel-
oping autophagosomes [98, 121]. HDAC6 is a micro-
tubule associated acetylase that binds to, and utilizes,
dynein motors to transport ubiquitinated proteins along
microtubles to a structure called the aggresome where
they are recognized by developing autophagosomes
[53]. As discussed below, the pattern of ubiquitina-
tion will determine whether the protein is targeted for
autophagic versus proteasomal degradation [98, 121].
The fusion step is mediated by dynein transpor-
tation of the autophagosomes along microtubules
to fuse with endosomes or lysomes [99]. Microtu-
bule disrupting agents, such as taxanes, vinblastine
or nocodazole, can prevent the fusion and cause
accumulation of autophagic vacuoles [32]. Inhibition
of lysosomal acidification by Bafilomycin A, a specific
V-ATPase (vacuolar H+ ATPase) inhibitor, or by other
lysosomal function inhibitors, CQ (Chloroquine) and
HCQ (hydroxychloroquine), also cause accumula-
tion of autophagosomes [122, 126]. Upon fusion with
the lysosome, the contents are degraded and the
components, including amino acids and lipids, are
released for re-utilization in cellular metabolism. The
TCA (tricarboxylic acid) cycle, which utilizes amino
acids for generation of bioenergetic molecules and
Fig. 2. Integration of macroautophagy with the regulatory network controlling the maintenance of cellular homeostasis versus
apoptosis. The Grp78 under ER stress has two effects, increased Grp78 expression induces autophagic vesicle formation while
release of Grp78 inhibition of PERK, EIF2α and ATF6 induce UPR. ATF4 is listed next to PERK because one study indicated that
ATF4 stability is responsible for the induction of autophagy. Arrows indicate induction and crossed lines indicate repression. Dashed
line indicate hypothesized, not proven, mechanism of Beclin 1 fragment induction of apoptosis. Large open arrow indicates that all
three components contribute to CHOP induction
Experimental Oncology 34, 286–297, 2012 (September)34, 286–297, 2012 (September) (September) 289
biosynthetic intermediates appears to co-ordinate with
macroautophagy through negative feedback of the
TCA substrate, pyruvate, on macroautophagy [81]. Al-
though not specific for macroautophagy, staining with
lysomotropic agents, such as acridine orange, is an ac-
ceptable marker for induction of macroautophagy [91].
Electron micrograph evidence of double-membraned
vesicles containing cytoplasmic components is a gold
standard for the presence of autophagosomes inside
cells, while cleared vesicles provide evidence that the
autophagic process is proceeding through to diges-
tion of the contents and not blocked by lysomotropic
agents or other situations. An example of electron
micrographic images of autophagosomes and cleared
autophagolysomes induced in human ovarian cancer
cells by treatment with a novel anti-cancer drug called
SHetA2 (NSC 726189) is shown in Fig. 3.
INTEGRATION OF THE PROTEASOMAL
SYSTEM AND AUTOPHAGY
The proteasome partners with autophagy to recycle
cellular proteins by digesting single soluble proteins
and releasing peptides into the cytoplasm and nucleus
to be digested by peptidases, or to be transferred
to the ER where they are bound by HLA (human leuko-
cyte antigen) proteins and eventually presented on the
surface of the cell [25, 64]. Proteasomes are multi-
protein complexes made up of an inner 20S cylindri-
cal shaped core of subunits that have the proteolytic
activity and a 19S regulatory cap of subunits on each
end of the core which recognize ubiquitin-conjugated
proteins and provide ATPase activity [36]. The protea-
some degrades type I (short-lived) and type II (mal-
folded/dysfunctional) proteins, whereas autophagy
degrades type II and type III (long-lived) proteins [14].
The cellular ubiquitination system specifies
whether a protein will be transferred to the protea-
some or an autophagosome for recycling. Proteins
with attached polyubiquitin chains that are branched
on Lys48 have a more closed conformation and are
targeted for proteasomal degradation, while proteins
with single ubiquitin moieties or polyubiquitin chains
that are branched on other Lys residues are targeted
to the autophagosomes as described above [98,
121]. Some proteins can be digested by both the
proteasome and autophagic vesicles [64]. Degrada-
tion of the proteins via the proteasome or autophagy
is ultimately determined through competition for bind-
ing by adapter proteins, p62 and NBR, which shuttle
the proteins to autophagic vesicles, or to p97, which
shuttles the proteins to the proteasome [98, 125]. The
p62 protein has a higher affinity for the monoubiquiti-
nated and Lys63 polyubiquitinated proteins targeted
for autophagic degradation, but can also recognize
Lys48 polyubiquitin chains targeted for proteasomal
degradation, suggesting that p62 can compensate for
loss of proteasomal function by bringing Lys48 ubiq-
uitylated proteins to the autophagosome when these
proteins accumulate during proteasome overload
or disfunction [40, 121].
There are several additional levels at which the pro-
teasome and autophagy are integrated. Inhibition of the
proteasome leads to induction of autophagy [25–27],
and induction of autophagy can protect cells from
death induced by proteasomal inhibitors [52]. On the
other hand, the proteasomal degradation pathway
does not appear to compensate for loss of autophagy.
Inhibition of early stages of autophagy results in buildup
of the p62 adapter, which brings proteins, which would
normally be degraded by the proteasome, instead
to be accumulated in aggresomes where they can-
not be accessed by the proteasomal machinery [63].
Inhibition of autophagy at that last stage of lysosomal
degradation by CQ, which does not cause aggresome
accumulation, but instead allows the buildup of the
ubiquitinated proteins inside lysosomal compartments,
also is not compensated by proteasome activity [11].
This unequal relationship between autophagy and pro-
teasomal degradation is highlighted by the observations
of proteasomes inside closed autophagic vesicles [21].
The induction of autophagy in response to proteasome
inhibition is mediated through the unfolded protein
response (UPR), which is induced when proteasomal
inhibitors cause accumulation of polyubiquitinated
proteins leading to ER stress (Fig. 2) [52].
INTEGRATION OF AUTOPHAGY WITH
ER STRESS AND UPR
ER stress is caused by buildup of unfolded, mis-
folded or damaged proteins that exceeds the capacity
of chaperones available in the ER to fold them. The UPR
sets off a series of events that mitigate this stress and
can also lead to induction of autophagy. The observa-
tion of ER stress, indicated by swollen ER, occurring
in the same cell as autophagic vesicles upon treatment
with the SHetA2 anticancer drug, but not upon treat-
ment with solvent only, supports the interconnection
of these two processes (Fig. 3). An excess of unfolded/
misfolded proteins interferes with the repressive effect
of the ER-resident chaperone, Grp78 (glucose regu-
lated protein 78, also called HSPA5 [heat shock protein
A5] or BiP [Binding immunoglobulin protein]) on three
UPR-regulatory proteins called PERK (double-stranded
RNA-dependent protein kinase (PKR)-like ER kinase),
IRE1α (inositol-requiring enzyme 1α) and ATF6 (Activat-
ing transcription factor 6) [93] (Fig. 2). Once released,
PERK, IRE1α and ATF6 work in concert to mediate the
UPR survival response involving arrest of general pro-
tein synthesis, while selected synthesis of chaperone
proteins is allowed to continue in order to restore the
balance of unfolded proteins/chaperones needed for
ER homeostasis [43]. To further reduce the ER load,
UPR can cause retrograde translocation of ER proteins
to the cytoplasm where they are degraded in the pro-
teasome through ERAD (ER associated degradation)
[84]. In situations of excess, irreparable or prolonged
stress, UPR can transition into apoptosis by upregula-
tion of CHOP (CCAAT/-enhancer- binding protein ho-
mologous protein) and downstream GADD34 (Growth
arrest and DNA damage 34), but in some situations
290 Experimental Oncology 34, 286–297, 2012 (September)
autophagy can intervene and prevent cell death by re-
moving the accumulated polyubiquitinated proteins and
aggregates [29, 90].
ER stress induces autophagy directly through
upregulation of Grp78 and through mechanisms
downstream of Grp78 release of the three UPR
signal transducers (Table 1, Fig. 2). A critical role
for Grp78 in the induction of autophagy was de-
monstrated with knockdown of Grp78 in normal
and cancer cells, which prevented autophagosome
formation in response to starvation or in response
to inhibition of protein processing with tunicamycin,
an inhibitor of N-linked glycosylation [74]. This study
also provided evidence for an integral co-dependency
of intact ER and autophagy. The massively dilated and
disrupted ER and the deficient autophagosome forma-
tion induced by Grp78 knockdown were both alleviated
by simultaneous knockdown of the XBP-1 transcription
factor, a downstream UPR mediator of IRE1α action
required for ER expansion [71], suggesting that intact
ER is maintained by and/or required for autophagy. The
link between Grp78 and autophagy induction occurs
downstream of nucleation, as the Grp78 knockdown
had no effect on Beclin 1/Vps34 association.
The link between the IRE1α arm and upregulation
of autophagy is mediated by IRE1α activation of JNK
(c-Jun N terminal kinase). Proteasomal inhibition
with bortezomib or MG132 induced ER stress and
autophagy in colon cancer cells, but not in the pres-
ence of siRNA reduction of IRE1α or chemical inhibition
of JNK activity, or in IRE1α knockout MEFs (murine em-
bryonic fibroblasts) [29]. In this study, the JNK inhibitor
had no effect on XBP-1 activation and was equally ef-
fective in XBP-1-positive and -negative cells indicating
that the UPR induction of autophagy occurs through
JNK and not the IRE1α arm of UPR. As discussed
above however, the XBP-1 function may be required
to maintain the ER in a functional state to support au-
tophagic vesicle formation. A similar study conducted
in neuroblastoma cells using specific siRNA knock-
down of IRE1α, PERK or ATF6, or using a JNK inhibitor,
demonstrated that ER stress up-regulated autophagy
through a mechanism dependent on IRE1α activation
of JNK, but independently of PERK and ATF6 [90].
The ER stress was induced with tunicamycin or thap-
sigargin, a chemical that blocks ER calcium uptake
by inhibiting ER Ca2+/ATPase. In this model, autophagy
protected against cell death as demonstrated by the
increase in cell death when autophagy was inhibited
by chemical (3-MA [3-methyladenine]) or genetic
manipulation (ATG7 siRNA), and inhibited cell death
when autophagy was induced by chemical stimulation
(rapamycin). The mechanism of autophagy induction
downstream of JNK appears to be a result of JNK phos-
phorylation of Bcl-2, which releases Bcl-2 repression
of Beclin mediation of autophagy [116].
Table 1. Mechanisms of ER stress and UPR induction of autophagy
Stressor Cell type Mechanism of autophagy
upregulation R
Grp78:
Tunicamycin,
Starvation
Embryonal kidney,
cervical cancer
Downstream of nucleation
(Vps34/Beclin1)
[74]
IRE1α Arm:
Bortezomib,
MG132
Colon cancer,
Prostate cancer
IRE1α → JNK → LC3B lipi-
dation
[29]
Tunicamycin,
Thapsigargin
Neuroblastoma IRE1α → JNK → LC3B lipida-
tion → autophagosomes
[90]
PERK Arm:
Misfolded poly-
glutamine re-
peats
Embryonal carci-
noma
PERK phosphorylation →
eIF2α phosphorylation →
LC3 lipidation (possibly through
increased ATF12 transcription)
[65]
Bortezomib Pancreatic cancer PERK phosphorylation →
eIF2α phosphorylation →
ATF4 → Atg 5 and Atg 7 trans-
cription
[129]
Bortezomib Breast cancer Reduction of proteasomal
degradation of ATF4 → LC3B
transcription
[85]
PERK, IRE1 α and ATF6 Arms:
SPP1 depletion Breast cancer ↑Grp78→ PERK+ IRE1α
+ATF6 → LC3B lipidation
[72]
Indirect:
BrefeldinA,
Thapsigargin,
Tunicamycin
Gingival fibroblast p38MAPK → ↑Grp78 and
↑Beclin1 and autophagic ves-
icles (possibly through p38 in-
hibition of mTORC1)
[55]
R = reference
Fig. 3. Simultaneous induction of ER stress and autophagy. Electron micrographs (X3250 magnification) of the human SK-OV-3 ovar-
ian cancer cell line treated with the SHetA2 anti-cancer drug (NSC 721689) for 17 hrs demonstrate normal nuclei (n) swollen ER
(SER) and normal ER, double-membraned autophagic vessicles (A) and autophagolysosomes (AL) cleared of their contents, while
control cells treated with the same volume of vehicle (dimethylsulfoxide) for 17 hrs do not exhibit SER, A or AL. The lack of clearly
identified mitochondria (M) in the treated cells suggests that they may have been digested by autophagy. The inset is enlarged
to show the double-membrane on an autophagosome
Experimental Oncology 34, 286–297, 2012 (September)34, 286–297, 2012 (September) (September) 291
The link between the PERK arm and upregulation
of autophagy is ultimately mediated by ATF4-driven
transcriptional upregulation of the ATG genes. Treat-
ment of embryonal carcinoma cells with misfolded
polyglutamine repeats caused buildup of polyubiq-
uitinated protein aggregates and induced LC3 lipi-
dation through a mechanism dependent on PERK
phosphorylation and activation of eIF2α, and result-
ing in autophagic elimination of the aggregates [65].
When the proteasomal inhibitor bortezomib was
used in pancreatic cancer cells, the eIF2α led to ac-
tivation of ATF4-driven transcription of the ATG5 and
ATG7 genes [129]. Activation of eIF2α also was shown
to mediate induction of autophagy by starvation and
viral infection [110]. Bortezomib treatment of breast
cancer cells led to reduction of proteasomal degrada-
tion of ATF4, thereby increasing ATF4-mediated LC3B
gene transcription [85].
All three of the UPR arms appear to be involved
in the induction of autophagy in breast cancer cells
caused by accumulation of the sphingolipid metabolite
S1P (sphingosine-1-phosphate) phosphatase [72].
In this study, S1P levels were increased by siRNA si-
lencing of the SPP1 (S1P phosphatase) responsible
for degradation of S1P in the ER. The resulting induc-
tion of autophagy was prevented by siRNA silencing
of PERK, IRE1α, or ATF6 or a dominant negative
PERK mutant. Other upstream inducers of Grp78 and
autophagy that have not been characterized for spe-
cific UPR arm involvement in the mechanism include
p38MAPK (p38 mitogen-activated protein kinase),
which likely induces autophagy through inhibition
of mTORC1 [55].
The ability of cellular UPR and autophagy respons-
es to ER stress to sufficiently mitigate the stress and
allow survival is dependent upon the transformation
state of the cell and the degree of stress. Although
autophagy was induced in both cancer and non-trans-
formed cell lines by ER stress inducers tunicamycin,
thapsigargin, A23187 (calcium ionophore) or brefeldin
A (protein transport inhibitor) and could reduce the
buildup of polyubiquitinated proteins, suppression
of autophagy using chemical (3-MA) or genetic manip-
ulation (siRNA to Beclin 1 or LC3B) reduced cell death
in colon and prostate cancer cell lines, but increased
cell death in normal non-transformed fibroblast and
non-immortalized human colon cell line [28]. The au-
thors of this study theorize that the macroautophagy
in non-cancer cells may lead to cell death by digest-
ing normal by-stander cellular constituents needed
for survival. Cancer cells characteristically become
growth-independent of these survival factors.
The switch between autophagy mediation of cell
survival versus death has been shown to be related
to the level of stress induced. In normal rat kidney cells,
autophagy induced in response to a low dose of cis-
platin was required for cell survival, while autophagy
induced in response to a high dose of cisplatin was
required for cell death [103]. ER stress and Grp78 ap-
pear to mediate the induction of autophagy in this
model. In a study of ER stress inducers (thapsigargin
and tunicamycin) in murine embryonal fibroblasts, low,
sublethal doses induced Grp78 expression and acti-
vated PERK, IRE1α and ATF6α [104]. Although CHOP
was also induced, the upregulation was lost within
24 hrs. Even when CHOP expression was persistently
up-regulated, only expression of the down-stream
GADD34 correlated with cell death. The authors of this
study conclude that differential stability of the Grp78,
CHOP and GADD34 mRNA’s and proteins contribute
to the ultimate fate of the cell.
Similar to the addiction of some cancer cells to cer-
tain oncogenes, cancer cells may become dependent
upon certain aspects of macroautophagy for cell sur-
vival and tumor growth, while defects in macroautoph-
agy that accumulate during tumor progression may
allow adaptations that prevent apoptosis despite the
presence of damaged proteins and organelles [118].
Consistent with this postulate, autophagy has been
shown to be critical for K-ras transformation of breast
cells [58]. In this model, K-ras induced transformation
through a mechanism involving ROS (reactive oxygen
species) induction of JNK. As discussed below, inhib-
iting autophagy is a current strategy in clinical trials
to enhance chemotherapeutic response and overcome
resistance of cancer cells.
AUTOPHAGY INTEGRATION WITH
APOPTOSIS
Macroautophagy has been reported to play a role
in cell death independently of apoptosis, but it remains
unclear if this is a consequence of the severity and/
or extended duration of autophagy, or as a deliberate
mechanism of programmed cell death. Autophagic
cell death is often described as cell death in the ab-
sence of apoptotic caspase activation and presence
of autophagic vesicles, but there lacks mechanism that
clearly defines autophagic cell death [37]. Whether
autophagy contributes to, or reduces, apoptosis
in cancer cells is dependent upon the type of cell
and the type and duration of stimulus. For instance,
autophagy can act as a either a survival or death
mechanism within the human SK-OV-3 ovarian cancer
cell line, depending on the molecule used to treat the
cells [62, 70, 128]. Also, loss of autophagy promotes
or prevents fibroblast apoptosis depending on the
death stimulus [115]. In the majority of oncology stud-
ies however, autophagy appears to play a protective
role. For example, in multiple myeloma cells, genetic
and chemical inhibition of autophagy enhances induc-
tion of apoptosis in vitro and tumor growth inhibition
by DNA-damaging drugs, doxorubicin and melphalan
in vivo [92]. In rat C6 glioma cells, siRNA silencing
of ATG5, ATG7 or ULK1 genes increased apopto-
sis caused by cyclosporine A [16]. A recent study
screened over 1400 cytotoxic agents for their ability
to induce autophagic cell death in the U2Os osteosar-
coma cell line [107]. Of the 59 compounds that were
validated to truly induce autophagic flux, none of them
292 Experimental Oncology 34, 286–297, 2012 (September)
were prevented from inducing cell death when the
ATG7 gene critical for autophagy was knocked out.
Some fragments of the autophagic machinery
are directly involved in the induction of apoptotic cell
death through the intrinsic mitochondrial pathway. The
transition to apoptosis appears to occur after autopha-
gy has been working to save the cell, but activation
of specific proteases cleave Atg5 or Beclin 1 releas-
ing cleavage products that translocate to the mito-
chondria and induce the intrinsic apoptosis pathway
(Fig. 2). This direct involvement of Atg5 in apoptosis
was demonstrated by enhanced sensitivity of multiple
cancer cell types to several cytotoxic chemotherapeu-
tic agents upon increased expression of Atg5 protein
in vitro and in vivo, while silencing of the ATG5 gene had
the opposite effect [127]. In this study, the switch from
macroautophagy to apoptosis was mediated by cal-
pain cleavage of the Atg5 protein, releasing a truncated
Atg5 that translocated to the mitochondria where
it bound the anti-apoptotic Bcl-2 molecule, BclXL,
thereby relieving BclXL inhibition of the BAX/BAK pore
forming ability resulting in cytochrome c release from
the mitochondria and caspase activation of apoptosis
[127]. The Bcl-2 family of proteins also integrates mac-
roautophagy and apoptosis through Beclin 1. Under
non-stressed conditions, Bcl-2 binds and sequesters
Beclin 1 resulting in suppression of autophagosome
formation [94]. Under stressed conditions, JNK be-
comes activated and phosphorylates Bcl-2, leading
to Bcl-2 degradation and release of Beclin 1 to allow
macroautophagy to attempt to recover cellular homeo-
stasis [116, 117]. Transition to apoptosis occurs when
caspase 3 is activated resulting in a Beclin 1 C-terminal
cleavage product that translocates to the mitochondria
and enhances apoptosis by releasing pro-apoptotic
factors [31, 120]. The known binding of Beclin 1 to the
anti-apoptotic BclXL protein is a possible explanation
for the induction of apoptosis, similar to what has been
reported for Atg5 [88].
Another level of integration between autophagy and
apoptosis is controlled by HMGB1 (high mobility group
box 1), a nonhistone DNA-binding protein that binds
tightly to chromatin of apoptotic cells and is released
from necrotic cells [2]. In response to autophagic
stimuli, HMGB1 translocates from the nucleus to the
cytosol where it binds directly to Beclin 1 causing the
release of Bcl-2 and allowing Beclin 1 to increase au-
tophagy [111]. Much less is known about the integra-
tion of autophagy and programmed necrosis. Although
autophagic vesicles have been observed in necrotic
cells [2], it is not clear if autophagy is contributing
to the cell death in this situation. Based on the ob-
servation that combined inhibition of autophagy and
apoptosis stimulates necrosis in cancer cells exposed
to ischemic conditions, autophagy appears to prevent
necrosis [24].
Inconsistent ordering of autophagy and UPR events
reported to occur prior to apoptosis suggest that the
transition from cell survival to apoptotic death is not
linear, but instead driven by an integrated network
of events that can eventually tip the balance to sur-
vival or death. For example, JNK can be activated
upstream of macroautophagy by ER stress, but can
also be activated downstream of macroautophagy
by etoposide or staurosporine treatment of apoptosis-
deficient MEFs [108]. This inconsistency suggests that
the pathways are not connected in a linear sequence,
but instead integrated at multiple levels. A simplified
interpretation may be that autophagic cell death is not
a mechanism designed by the cell, but rather a rare
consequence of too much autophagy in response
to external stimuli, such as chemical reagents or pro-
longed stress. The unrestrained autophagy could lead
to cell death in the absence of apoptosis by consum-
ing the viable cell mass or by upsetting the balance
of pro-survival versus pro-apoptotic proteins. This may
explain why a definitive molecular mechanism for pro-
grammed autophagic cell death has not been defined.
TARGETING MACROAUTOPHAGY
IN CANCER THERAPY
While there is evidence that macroautophagy can
suppress the early development of cancer [13], upreg-
ulation of basal levels of autophagy in multiple cancers
indicate that autophagy primarily drives survival once
the tumor has formed [33]. A recently developed meth-
od to detect macroautophagy in clinical specimens
using immunohistochemistry with an antibody to LC3B
demonstrated that punctuate pattern staining of LC3B
indicative of macroautophagy significantly correlated
with heightened cell proliferation and nuclear grade,
invasion and metastasis, and worse outcome [69].
This is of particular significance because multiple anti-
cancer agents are known to induce macroautophagy,
which could interfere with tumor response (Table 2).
Table 2. Anti-cancer agents that induce autophagy
Function Name R
Alkylating agents Cyclophosphamide, Temozolomide [3, 50]
Bcl2 inhibitor GX15-070 [47]*
Farnesyltransferase in-
hibitor
Lonafarnib [47]*
Glycolysis inhibitors GX15-070, 2-deoxyglucose [47]*
HDAC inhibitors Vorinostat, Sodium butyrate, LAQ824,
Panobinostat
[47]*
Hormone treatments Tamoxifen,Toremifene [47]*
Ionizing radiation Cs-137 [91,124]
Microtubule inhibitor Vinblastine [101]
Monoclonal antibodies Rituximab (to CD20), Panitumumab
(to EGF-R)
[47]*
mTOR inhibitors Sirolimus, Temsirolimus, Everolimus,
NV-128
[47]*
Natural compounds Arsenic, resveratrol [47]*
PARP inhibitor ABT-888 [47]*
Proteasome inhibitors Bortezomib, NPI-0052114, Epoxomicin [47]*
Topoisomerase poisons Doxorubicin, Campothecin [73, 78]
Tyrosine kinase inhibitors Dasatinib, Sorafenib, Imatinib [47]*
Vitamin D analog EB1089 [47]*
R = references, * Multiple references are provided in this review.
Preclinical studies demonstrating a protective role
for macroautophagy in response to these therapeutics
have translated into multiple clinical trials evalua ting
CQ and the derivative HCQ (hydroxychloroquine)
inhibition of autophagy in combination with a variety
of current cancer treatment strategies. CQ and HCQ
are “old drugs” that have been prescribed for malaria
Experimental Oncology 34, 286–297, 2012 (September)34, 286–297, 2012 (September) (September) 293
[89], rheumatoid arthritis [66] and HIV [102]. The first
reported clinical trial of combining autophagy inhibition
with cancer therapy was a single-institution phase III
trial in glioblastoma patients treated with conventional
radiation and carmustine therapy with or without daily
CQ [109]. Although this study was not adequately
powered to detect a significant difference in survival,
CQ increased overall survival from 11 months in the
placebo arm to 24 months in the CQ arm. HCQ is a less
toxic version of CQ and the best autophagy inhibitor
currently commercially available for clinical trials [39].
Currently there are 84 clinical trials of HCQ listed on the
United States Government website (ClinicalTrials.gov),
of which 33 are cancer studies, including 15 Phase I,
nine Phase I/II, and nine Phase II clinical trials of HCQ
in combination with a range of chemotherapeutic
agents.
In contrast to the sensitization of tumors to che-
motherapeutic agents by autophagy inhibition, the
sensitivity of tumors to radiation has been shown
to be enhanced by induction of autophagy with the
mTOR inhibitor everolimus (RAD001) and further
enhanced by combined inhibition of apoptosis and
induction of autophagy [9, 57]. There are several early
phase clinical trials of everolimus or rapamycin in com-
bination with radiation therapy for a variety of cancers
listed on the ClinicalTrials.gov website. The translation
of laboratory-based autophagy studies to clinical trials
is based primarily on research of macroautophagy,
however there is considerable integration of mac-
roautophagy with the other forms of autophagy that
could be taken advantage of in this cancer treatment
strategy, and that needs to be taken into consideration
to fully understand the impact of the interventions and
the interpretation of results.
MECHANISM AND INTEGRATION
OF CHAPERONE-MEDIATED AUTOPHAGY
CMA involves the selective degradation of indi-
vidual molecules containing an amino acid sequence
motif related to KFERQ (Lys-Phe-Glu-Arg-Gln) [27].
CMA activity can be measured in cells by the transition
of diffuse to punctuate pattern of a photoconvertible
KFERQ-PA-mCherryl reporter protein in transfected
cells [61]. It is estimated that at least 30% of cytosolic
proteins contain this sequence or a sequence that can
be made mimic KFERQ through post-translational
modifications [26]. The proteins may have KFERQ lo-
cated on their surface for recognition by Hsc70 or this
sequence may become exposed upon damage or de-
naturing of the protein, or upon separation of the pro-
tein with another protein subunit. A single molecular
chaperone (Hsc70/HSPA8) appears to mediate the
recognition of proteins with exposed KFERQ domains
and transporting them to the lysosome [15]. At the
lysosome, Hsc70 binds LAMP-2A (Lysosome-asso-
ciated membrane protein type 2A), and interacts with
a complex of other chaperones to unfold the trans-
ported protein and push it through LAMP-2A to an-
other molecule of Hsc70 waiting inside the lysosome
[1]. Increased expression of LAMP2A and lysosomal
Hsc70 correlate with CMA activity and are accepted
markers of CMA [18, 19]. Hsc70 binds and hydrolyzes
ATP in order to generate the energy required for this
process, and the ADP-bound form of Hsc70 has the
highest affinity for KFERQ proteins [48]. The proteins
working with Hsc70 at the surface of the lysosome
have unique functions. Hsp40 activates Hsc70 ATPase
activity to increase affinity for protein substrates, Hip
(heat shock protein 70 interacting protein) facilitates
the assembly of the various proteins in the complex,
cdc48 (cell division cycle 48) stimulates the activity
of this protein complex, and Bag-1 (Bcl-2-associated
athanogene 1) acts as a nucleotide exchange factor
that stimulates substrate release [76]. The stability
of Hsc70 inside the lysosome is tightly controlled by the
pH of the lumen, which, if altered, could denature
Hsc70 and make it susceptible to lysosomal proteases
[19]. LAMP-2A also works as a complex with EF1α
(elongation factor 1 α) to form a complex with GFAP
(glial fibrillary acidic protein) that can be negatively
regulated by GTP, which causes dissociation of the
complex subunits [5].
CMA is a second line response to cell starvation [7].
Macroautophagy is the first line response to nutrient
deprivation with maximal activity around 6 hours, fol-
lowed by a gradual reduction [35, 83]. There appears
to be mutual inhibition between macroautophagy and
CMA, as CMA does not increase to suprabasal levels
until the reduction of macroautophagy 6 to 8 hours
after initiation of starvation [7]. Maximal CMA occurs
24 hours after initiation of starvation and continues
for at least 3 days [20]. Although inhibition of macro-
autophagy or CMA can lead to upregulation of each
other, the compensation is incomplete, as CMA cannot
degrade organelles and macroautophagy cannot com-
pensate for the selectivity of CMA. Inhibition of CMA
by blocking expression of LAMP-2A in fibroblasts
resulted in increased macroautophagy, however this
compensatory action did not alleviate the increased
sensitivity of CMA-deficient cells to stress [83]. In-
hibition of macroautophagy with type III PI3K inhibi-
tors (3-3-MA, wortmannin or LY294002) or activation
of macroautophagy with rapamycin, had no effect
on CMA [35], however macroautophagy-deficient
cells caused by genetic deletion of ATG5 exhibit up-
regulation of both basal and induced CMA through
different mechanisms [51]. Although there is little
evidence to document direct integration of CMA with
ER stress or UPR, integration of CMA with proteasomal
degradation is indicated by the selective degradation
of proteasomal catalytic core subunits by CMA [21].
Deregulated CMA has been shown to cause mul-
tiple diseases including MLIV (mucolipidosis Type
IV), which is caused by defects in TRPML1 (transient
receptor potential mucolipin-1) leading to ineffec-
tive docking with Hsc70 inside the lysosome [114],
glaucoma and other diseases related to ineffective
Hsc70 transport of proteins along neuronal axons [39].
In cancer, levels of the MDM2 (murine double minute
294 Experimental Oncology 34, 286–297, 2012 (September)
2) regulator of the p53 tumor suppressor protein and
the Gal3 (Galectin-3) oncogenic protein, and likely
multiple other oncongenes and tumor suppressor
genes, are controlled by CMA [77, 80]. A recent study
demonstrated that inhibition of constitutively upregu-
lated CMA by silencing Lamp-2A expression caused
increased p53 levels resulting in reduced proliferation
and altered metabolism in human lung cancer cells
in vitro and reduced tumor growth and metastases
in vivo, indicating that CMA is a relevant target for
anti-cancer therapy [61].
MICROAUTOPHAGY
Although microautophagy was originally described
by de Duve and Wattiauz in 1963, the term was not
coined until 1983 [23]. While much less well chara-
cterized than macroautophagy and CMA, studies
of microautophagy have led to the delineation of 5 se-
quential steps that mediate microautophagy, namely:
invagination, vesicle formation, vesicle expansion,
vesicle scission and vesicle degradation and recycling
[75]. The first step of invagination is an ATP-depen-
dent process that occurs at areas of the lysosomal
membrane with low concentrations of transmem-
brane proteins and that develops as tubes filled with
cytoplasmic components, in contrast to the shapes
of other types of lysosomal invaginations [82, 106].
The subsequent processes are mediated by mTOR
regulated ubiquitin-like systems, LC3 lipidation and
other Atg machinery similar to that discussed above
for macroautophagy [75]. Selective microautophagy
degradation of mitochondria (micromitophagy), the
nucleus (PMN [piecemeal microautophagy of the nu-
cleus]) and peroxisomes (micropexophagy) have been
described [34, 54, 67]. Similar to macroautophagy
and CMA, microautophagy occurs at basal levels and
can be induced by nitrogen starvation or mitochon-
drial damage, however in contrast to the antagonism
of macroautophagy with CMA, microautophagy ap-
pears to work synergistically with the other two forms
of autophagy [17]. Although primarily characterized
in yeast, defects in microautophagy have been linked
to a number of human diseases, however a direct link
with cancer has not been identified [75].
CONCLUSIONS
Are current definitions of pathways and modes
of cell stress response, survival and death limiting our
ability to comprehend the dynamic cell? Models are
useful, but can introduce bias into our comprehension.
This review has described multiple levels of integration
between various pathways that can maintain cellular
homeostasis or default into apoptosis. Upon stress
to the system, the ultimate consequence of cell sur-
vival or death depends on balance of events linked
at multiple network connections, similar to a hanging
mobile. In this model, failure to balance the weights
of proteasomal degradation, UPR and autophagy
against apoptosis and other forms of cell death, such
as programmed necrosis, can lead to excessive au-
tophagy consuming the viable cell mass or to the relief
of inhibition of the default apoptosis pathway that is al-
ways present and waiting for relief of inhibition to kill
the cell. The detailed molecular interactions described
in this review provide information on the nodes of this
complex network that can be manipulated to control
the network in development of strategies to induce
apoptosis in cancer cells without harming normal cells.
REFERENCES
1. Agarraberes FA, Terlecky SR, Dice JF. An intralyso-
somal hsp70 is required for a selective pathway of lysosomal
protein degradation. J Cell Biol 1997; 137: 825–34.
2. Amaravadi RK, Thompson CB. The roles of therapy-
induced autophagy and necrosis in cancer treatment. Clin
Cancer Res 2007; 13: 7271–9.
3. Amaravadi RK, Yu D, Lum JJ, et al. Autophagy inhibi-
tion enhances therapy-induced apoptosis in a Myc-induced
model of lymphoma. J Clin Invest 2007; 117: 326–36.
4. Avruch J, Hara K, Lin Y, et al. Insulin and amino-acid
regulation of mTOR signaling and kinase activity through the
Rheb GTPase. Oncogene 2006; 25: 6361–72.
5. Bandyopadhyay U, Sridhar S, Kaushik S, et al. Identi-
fication of regulators of chaperone-mediated autophagy. Mol
Cell 2010; 39: 535–47.
6. Barth S, Glick D, Macleod KF. Autophagy: assays and
artifacts. J Pathol 2010; 221: 117–24.
7. Bejarano E, Cuervo AM. Chaperone-mediated au-
tophagy. Proc Am Thorac Soc 2010; 7: 29–39.
8. Benbrook DM, Masamha CP. The pro-survival function
of Akt kinase can be overridden or altered to contribute to induc-
tion of apoptosis. Curr Cancer Drug Targets 2011; 11: 586–99.
9. Cao C, Subhawong T, Albert JM, et al. Inhibition
of mammalian target of rapamycin or apoptotic pathway
induces autophagy and radiosensitizes PTEN null prostate
cancer cells. Cancer Res 2006; 66: 10040–7.
10. Carew J, Kelly K, Nawrocki S. Mechanisms of mTOR in-
hibitor resistance in cancer therapy. Target Oncol 2011; 6: 17–27.
11. Carew JS, Medina EC, Esquivel JA 2nd, et al. Autophagy
inhibition enhances vorinostat-induced apoptosis via ubiquiti-
nated protein accumulation. J Cell Mol Med 2010; 14: 2448–59.
12. Chan EY. mTORC1 phosphorylates the ULK1-mAtg13-
FIP200 autophagy regulatory complex. Sci Signal 2009; 2: pe51.
13. Chen N, Karantza-Wadsworth V. Role and regula-
tion of autophagy in cancer. Biochim Biophys Acta 2009;
1793: 1516–23.
14. Chen X, Yin XM. Coordination of autophagy and the
proteasome in resolving endoplasmic reticulum stress. Vet
Pathol 2011; 48: 245–53.
15. Chiang HL, Terlecky SR, Plant CP, Dice JF. A role for
a 70-kilodalton heat shock protein in lysosomal degradation
of intracellular proteins. Science 1989; 246: 382–5.
16. Ciechomska IA, Gabrusiewicz K, Szczepankiewicz AA,
Kaminska B. Endoplasmic reticulum stress triggers autophagy
in malignant glioma cells undergoing cyclosporine A-induced
cell death. Oncogene 2012 [Epub ahead of print].
17. Cuervo AM. Autophagy: many paths to the same end.
Mol Cell Biochem 2004; 263: 55–72.
18. Cuervo AM, Dice JF. Regulation of lamp2a levels in the
lysosomal membrane. Traffic 2000; 1: 570–83.
19. Cuervo AM, Dice JF, Knecht E. A population of rat liver
lysosomes responsible for the selective uptake and degradation
of cytosolic proteins. J Biol Chem 1997; 272: 5606–15.
20. Cuervo AM, Knecht E, Terlecky SR, Dice JF. Activa-
tion of a selective pathway of lysosomal proteolysis in rat liver
by prolonged starvation. Am J Physiol 1995; 269: C1200–8.
Experimental Oncology 34, 286–297, 2012 (September)34, 286–297, 2012 (September) (September) 295
21. Cuervo AM, Palmer A, Rivett AJ, Knecht E. Degrada-
tion of proteasomes by lysosomes in rat liver. Eur J Biochem
1995; 227: 792–800.
22. de Duve C. The lysosome. Sci Am 1963; 208: 64–72.
23. de Duve C, Wattiauz R. Function of lysosomes. Annu
Rev Physiol 1966; 28: 435–92.
24. Degenhardt K, Mathew R, Beaudoin B, et al. Autophagy
promotes tumor cell survival and restricts necrosis, inflamma-
tion, and tumorigenesis. Cancer Cell 2006; 10: 51–64.
25. Dengjel J, Schoor O, Fischer R, et al. Autophagy pro-
motes MHC class II presentation of peptides from intracellular
source proteins. Proc Natl Acad Sci USA 2005; 102: 7922–7.
26. Dice JF. Chaperone-mediated autophagy. Autophagy
2007; 3: 295–9.
27. Dice JF, Chiang HL, Spencer EP, Backer JM. Regula-
tion of catabolism of microinjected ribonuclease A. Identifi-
cation of residues 7–11 as the essential pentapeptide. J Biol
Chem 1986; 257: 14624–7.
28. Ding W-X, Ni H-M, Gao W, et al. Differential effects
of endoplasmic reticulum stress-induced autophagy on sell
survival. J Biol Chem 2007; 282: 4702–10.
29. Ding W-X, Ni H-M, Gao W, et al. Linking of autophagy
to ubiquitin-proteasome system is important for the regulation
of endoplasmic reticulum stress and cell viability. Am J Pathol
2007; 171: 513–24.
30. Dixon SJ, Lemberg KM, Lamprecht MR, et al. Fer-
roptosis: an iron-dependent form of nonapoptotic cell death.
Cell 2012; 149: 1060–72.
31. Djavaheri-Mergny M, Maiuri MC, Kroemer G. Cross
talk between apoptosis and autophagy by caspase-mediated
cleavage of Beclin 1. Oncogene 2010; 29: 1717–9.
32. Eskelinen E-L. Maturation of autophagic vacuoles
in mammalian cells. Autophagy 2005; 1: 1–10.
33. Espina V, Mariani BD, Gallagher RI, et al. Malignant
precursor cells pre-exist in human breast DCIS and require
autophagy for survival. PLoS One 2010; 5: e10240.
34. Farré J-C, Subramani S. Peroxisome turnover by mi-
cropexophagy: an autophagy-related process. Trends Cell Biol
2004; 14: 515–23.
35. Finn PF, Mesires NT, Vine M, Dice JF. Effects of small
molecules on chaperone-mediated autophagy. Autophagy
2005; 1: 141–5.
36. Frankland-Searby S, Bhaumik SR. The 26S protea-
some complex: an attractive target for cancer therapy. Biochim
Biophys Acta 2012; 1825: 64–76.
37. Galluzzi L, Vitale I, Abrams JM, et al. Molecular
definitions of cell death subroutines: recommendations of the
Nomenclature Committee on Cell Death 2012. Cell Death
Differ 2012; 19: 107–20.
38. Ganley IG, Lam du H, Wang J, et al. ULK1.ATG13.FIP200 com-
plex mediates mTOR signaling and is essential for autophagy. J Biol
Chem 2009; 284: 12297–305.
39. Garber K. Inducing indigestion: companies embrace
autophagy inhibitors. J Natl Cancer Inst 2011; 9: 708–10.
40. Geetha T, Seibenhener ML, Chen L, et al. p62 serves
as a shuttling factor for TrkA interaction with the proteasome.
Biochem Biophys Res Commun 2008; 374: 33–7.
41. Hailey DW, Rambold AS, Satpute-Krishnan P, et al.
Mitochondria supply membranes for autophagosome biogen-
esis during starvation. Cell 2010; 141: 656–67.
42. Hayashi-Nishino M, Fujita N, Noda T, et al. A subdo-
main of the endoplasmic reticulum forms a cradle for autopha-
gosome formation. Nat Cell Biol 2009; 11: 1433–7.
43. Hetz C. The unfolded protein response: controlling
cell fate decisions under ER stress and beyond. Nat Rev Mol
Cell Biol 2012; 13: 89–102.
44. Hosokawa N, Hara T, Kaizuka T, et al. Nutrient-depen-
dent mTORC1 association with the ULK1-Atg13-FIP200 com-
plex required for autophagy. Mol Biol Cell 2009; 20: 1981–91.
45. Ichimura Y, Kirisako T, Takao T, et al. A ubiquitin-like
system mediates protein lipidation. Nature 2000; 408: 488–92.
46. Inoki K, Kim J, Guan K-L. AMPK and mTOR in cellu-
lar energy homeostasis and drug targets. Annu Rev Pharmacol
Toxicol 2012; 52: 381–400.
47. Janku F, McConkey DJ, Hong DS, Kurzrock R. Au-
tophagy as a target for anticancer therapy. Nat Rev Clin Oncol
2011; 8: 528–39.
48. Jiang J, Prasad K, Lafer EM, Sousa R. Structural basis
of interdomain communication in the Hsc70 chaperone. Mol
Cell 2005; 20: 513–24.
49. Jung CH, Jun CB, Ro S-H, et al. ULK-Atg13-
FIP200 complexes mediate mTOR signaling to the autophagy
machinery. Mol Biol Cell 2009; 20: 1992–2003.
50. Kanzawa T, Germano IM, Komata T, et al. Role of au-
tophagy in temozolomide-induced cytotoxicity for malignant
glioma cells. Cell Death Differ 2004; 11: 448–57.
51. Kaushik S, Massey AC, Mizushima N, Cuer-
vo AM. Constitutive activation of chaperone-mediated
autophagy in cells with impaired macroautophagy. Mol Biol
Cell 2008; 19: 2179–92.
52. Kawaguchi T, Miyazawa K, Moriya S, et al. Combined
treatment with bortezomib plus bafilomycin A1 enhances the
cytocidal effect and induces endoplasmic reticulum stress
in U266 myeloma cells: crosstalk among proteasome, autoph-
agy-lysosome and ER stress. Int J Oncol 2011; 38: 643–54.
53. Kawaguchi Y, Kovacs JJ, McLaurin A, et al. The deacety-
lase HDAC6 regulates aggresome formation and cell viability
in response to misfolded protein stress. Cell 2003; 115: 727–38.
54. Kissová I, Salin B, Schaeffer J, et al. Selective and
non-selective autophagic degradation of mitochondria in yeast.
Autophagy 2007; 3: 329–36.
55. Kim D-S, Kim J-H, Lee G-H, et al. p38 Mitogen-
activated protein kinase is involved in endoplasmic reticulum
stress-induced cell death and autophagy in human gingival
fibroblasts. Biol Pharm Bull 2010; 33: 545–9.
56. Kim E, Goraksha-Hicks P, Li L, et al. Regulation
of TORC1 by Rag GTPases in nutrient response. Nat Cell
Biol 2008; 10: 935–45.
57. Kim KW, Hwang M, Moretti L, et al. Autophagy
upregulation by inhibitors of caspase-3 and mTOR enhances
radiotherapy in a mouse model of lung cancer. Autophagy
2008; 4: 659–68.
58. Kim M-J, Woo S-J, Yoon C-H, et al. Involvement
of autophagy in oncogenic K-Ras-induced malignant cell
transformation. J Biol Chem 2011; 286: 12924–32.
59. Klionsky DJ. Autophagy revisited: A conversation with
Christian de Duve. Autophagy 2008; 4: 740–3.
60. Klionsky DJ, Cregg JM, Dunn WA Jr, et al. A unified
nomenclature for yeast autophagy-related genes. Dev Cell
2003; 5: 539–45.
61. Kon M, Kiffin R, Koga H, et al. Chaperone-mediated
autophagy is required for tumor growth. Sci Transl Med 2012;
3: 109ra117.
62. Kong D, Ma S, Liang B, et al. The different regulatory
effects of p53 status on multidrug resistance are determined
by autophagy in ovarian cancer cells. Biomed Pharmacother
2012; 66: 271–8.
63. Korolchuk VI, Mansilla A, Menzies FM, Rubinsz-
tein DC. Autophagy inhibition compromises degradation
of ubiquitin-proteasome pathway substrates. Mol Cell 2009;
33: 517–27.
296 Experimental Oncology 34, 286–297, 2012 (September)
64. Korolchuk VI, Menzies FM, Rubinsztein DC. Mecha-
nisms of cross-talk between the ubiquitin-proteasome and
autophagy-lysosome systems. FEBS Lett 2010; 584: 1393–8.
65. Kouroku Y, Fujita E, Tanida I, et al. ER stress (PERK/
eIF2 alpha phosphorylation) mediates the polyglutamine-
induced LC3 conversion, an essential step for autophagy
formation. Cell Death Differ 2007; 14: 230–9.
66. Kremer JM. Rational use of new and existing disease-
modifying agents in rheumatoid arthritis. Ann Intern Med
2001; 134: 695–706.
67. Krick R, Mühe Y, Prick T, et al. Piecemeal microau-
tophagy of the nucleus: Genetic and morphological traits.
Autophagy 2009; 5: 270–2.
68. Kroemer G, Galluzzi L, Vandenabeele P, et al. and
Nomenclature Committee on Cell Death. Classification of cell
death: recommendations of the Nomenclature Committee
on Cell Death 2009. Cell Death Differ 2009; 16: 3–11.
69. Lazova R, Camp RL, Klump V, et al. Punctate LC3B
expression is a common feature of solid tumors and associated
with proliferation, metastasis, and poor outcome. Clin Cancer
Res 2012; 18: 370–9.
70. Le X-F, Mao W, Lu Z, et al. Dasatinib induces au-
tophagic cell death in human ovarian cancer. Cancer 2010;
116: 4980–90.
71. Lee AH, Chu GC, Iwakoshi NN, Glim cher LH. XBP-1 is re-
quired for biogenesis of cellular secretory machinery of exocrine
glands. EMBO J 2005; 24: 4368–80.
72. Lepine S, Allegood JC, Park M, et al. Sphingosine-
1-phosphate phosphohydrolase-1 regulates ER stress-induced
autophagy. Cell Death Differ 2011; 18: 350–61.
73. Li H, Wang P, Sun Q, et al. Following cytochrome
c release, autophagy is inhibited during chemotherapy-induced
apoptosis by caspase 8-mediated cleavage of Beclin 1. Cancer
Res 2011; 71: 3625–34.
74. Li J, Ni M, Lee B, et al. The unfolded protein response
regulator GRP78/BiP is required for endoplasmic reticulum
integrity and stress-induced autophagy in mammalian cells.
Cell Death Differ 2008; 15: 1460–71.
75. Li WW, Li J, Bao JK. Microautophagy: lesser-known
self-eating. Cell Mol Life Sci 2012; 69: 1125–36.
76. Li W, Yang Q, Mao Z. Chaperone-mediated auto-
phagy: machinery, regulation and biological consequences.
Cell Mol Life Sci 2011; 68: 749–63.
77. Li X, Ma Q, Wang J, et al. c-Abl and Arg tyrosine
kinases regulate lysosomal degradation of the oncoprotein
Galectin-3. Cell Death Differ 2010; 17: 1277–87.
78. Lin C-I, Whang EE, Abramson MA, et al. Autopha-
gy: a new target for advanced papillary thyroid cancer therapy.
Surgery 2009; 146: 1208–14.
79. Lindmo K, Stenmark H. Regulation of membrane traffic
by phosphoinositide 3-kinases. J Cell Sci 2006; 119: 605–14.
80. Lu T-L, Huang G-J, Wang H-J, et al. Hispolon pro-
motes MDM2 downregulation through chaperone-mediated
autophagy. Biochem Biophys Res Commun 2010; 398: 26–31.
81. Lum JJ, DeBerardinis RJ, Thompson CB. Autophagy
in metazoans: cell survival in the land of plenty. Nat Rev Mol
Cell Biol 2005; 6: 439–48.
82. Müller O, Sattler T, Flötenmeyer M, et al. Autophagic
tubes. The J Cell Biol 2000; 151: 519–28.
83. Massey AC, Kaushik S, Sovak G, et al. Consequences
of the selective blockage of chaperone-mediated autophagy.
Proc Natl Acad Sci USA 2006; 103: 5805–10.
84. Meusser B, Hirsch C, Jarosch E, Sommer T. ERAD: the
long road to destruction. Nat Cell Biol 2005; 7: 766–72.
85. Milani M, Rzymski T, Mellor HR, et al. The role
of ATF4 stabilization and autophagy in resistance of breast
cancer cells treated with Bortezomib. Cancer Res 2009;
69: 4415–23.
86. Mizushima N, Yamamoto A, Hatano M, et al. Dissec-
tion of autophagosome formation using Apg5-deficient mouse
embryonic stem cells. J Cell Biol 2001; 152: 657–68.
87. Nishida Y, Arakawa S, Fujitani K, et al. Discovery
of Atg5/Atg7-independent alternative macroautophagy. Na-
ture 2009; 461: 654–8.
88. Noble CG, Dong J-M, Manser E, Song H. BclXL and
UVRAG cause a monomer-dimer switch in Beclin1. J Biol
Chem 2008; 283: 26274–82.
89. O’Neill PM, Bray PG, Hawley SR, et al. 4-Amino-
quinolines — Past, present, and future: A chemical perspective.
Pharmacol Ther 1998; 77: 29–58.
90. Ogata M, Hino S, Saito A, et al. Autophagy is activated
for cell survival after endoplasmic reticulum stress. Mol Cell
Biol 2006; 26: 9220–31.
91. Paglin S, Hollister T, Delohery T, et al. A novel response
of cancer cells to radiation involves autophagy and formation
of acidic vesicles. Cancer Res 2001; 61: 439–44.
92. Pan Y, Gao Y, Chen L, et al. Targeting autophagy
augments in vitro and in vivo antimyeloma activity of DNA-
damaging chemotherapy. Clin Cancer Res 2011; 17: 3248–58.
93. Parmar VM, Schroder M. Sensing endoplasmic reticu-
lum stress. Adv Exp Med Biol 2012; 738: 153–68.
94. Pattingre S, Tassa A, Qu X, et al. Bcl-2 antiapoptotic
proteins inhibit Beclin 1-dependent autophagy. Cell 2005;
122: 927–39.
95. Petiot A, Ogier-Denis E, Blommaart EF, et al. Distinct
classes of phosphatidylinositol 3’-kinases are involved in sig-
naling pathways that control macroautophagy in HT-29 cells.
J Biol Chem 2000; 275: 992–8.
96. Pickart CM. Mechanisms underlying ubiquitination.
Ann Rev Biochem 2001; 70: 503–33.
97. Polson HE, de Lartigue J, Rigden DJ, et al. Mammalian
Atg18 (WIPI2) localizes to omegasome-anchored phago phores and
positively regulates LC3 lipidation. Autophagy 2010; 6: 506–22.
98. Popovic D, Dikic I. The molecular basis of selective
autophagy. Biochemist 2012; 34: 24–30.
99. Ravikumar B, Acevedo-Arozena A, Imarisio S, et al.
Dynein mutations impair autophagic clearance of aggregate-
prone proteins. Nat Genet 2005; 37: 771–6.
100. Ravikumar B, Moreau K, Jahreiss L, et al. Plasma
membrane contributes to the formation of pre-autophago-
somal structures. Nat Cell Biol 2010; 12: 747–57.
101. Rez G, Csak J, Fellinger E, et al. Time course of vinblas-
tine-induced autophagocytosis and changes in the endoplasmic
reticulum in murine pancreatic acinar cells: a morphometric and
biochemical study. Eur J Cell Biol 1996; 71: 341–50.
102. Romanelli F, Smith KM, Hoven AD. Chloroquine and
hydroxychloroquine as inhibitors of human immunodeficiency
virus (HIV-1) activity Curr Pharm Des 2004; 10: 2643–8.
103. Rovetta F, Stacchiotti A, Consiglio A, et al. ER sig-
naling regulation drives the switch between autophagy and
apoptosis in NRK-52E cells exposed to cisplatin. Exp Cell
Res 2012; 318: 238–50.
104. Rutkowski DT, Arnold SM, Miller CN, et al. Adapta-
tion to ER stress is mediated by differential stabilities of pro-
survival and pro-apoptotic mRNAs and proteins. PLoS Biol
2006; 4: e374.
105. Sancak Y, Peterson TR, Shaul YD, et al. The Rag
GTPases bind Raptor and mediate amino acid signaling
to mTORC1. Science 2008; 320: 1496–501.
106. Sattler T, Mayer A. Cell-free reconstitution of micro-
autophagic vacuole invagination and vesicle formation. J Cell
Biol 2000; 151: 529–38.
Experimental Oncology 34, 286–297, 2012 (September)34, 286–297, 2012 (September) (September) 297
107. Shen S, Kepp O, Michaud M, et al. Association and
dissociation of autophagy, apoptosis and necrosis by systematic
chemical study. Oncogene 2011; 30: 4544–56.
108. Shimizu S, Konishi A, Nishida Y, et al. Involvement
of JNK in the regulation of autophagic cell death. Oncogene
2010; 29: 2070–82.
109. Sotelo J, Briceño E, López-González MA. Adding
chloroquine to conventional treatment for glioblastoma mul-
tiforme. Ann Intern Med 2006; 144: 337–43.
110. Tallóczy Z, Jiang W, Virgin HW 4th, et al. Regulation
of starvation- and virus-induced autophagy by the eIF2alpha
kinase signaling pathway. Proc Natl Acad Sci USA 2002;
99: 190–5.
111. Tang D, Kang R, Livesey KM, et al. Endogenous
HMGB1 regulates autophagy. J Cell Biol 2010; 190: 881–92.
112. Tanida I. Autophagy basics. Microbiol Immunol
2011; 55: 1–11.
113. Vance JE, Vance DE. Phospholipid biosynthesis
in mammalian cells. Biochem Cell Biol 2004; 82: 113–28.
114. Venugopal B, Mesires NT, Kennedy JC, et al. Chape-
rone-mediated autophagy is defective in mucolipidosis type
IV. J Cell Physiol 2009; 219: 344–53.
115. Wang Y, Singh R, Massey AC, et al. Loss of macroau-
tophagy promotes or prevents fibroblast apoptosis depending
on the death stimulus. J Biol Chem 2008; 283: 4766–77.
116. Wei Y, Pattingre S, Sinha S, et al. JNK1-mediated
phosphorylation of Bcl-2 regulates starvation-induced au-
tophagy. Mol Cell 2008; 30: 678–88.
117. Wei Y, Sinha S, Levine B. Dual role of JNK1-mediated
phosphorylation of Bcl-2 in autophagy and apoptosis regula-
tion. Autophagy 2008; 4: 949–51.
118. Weinstein IB, Joe A. Oncogene addiction. Cancer Res
2008; 68: 3077–80.
119. Welchman RL, Gordon C, Mayer RJ. Ubiquitin and
ubiquitin-like proteins as multifunctional signals. Nat Rev Mol
Cell Biol 2005; 6: 599–609.
120. Wirawan E, Vande Walle L, Kersse K, et al. Caspase-me-
diated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy
and enhances apoptosis by promoting the release of proapoptotic
factors from mitochondria. Cell Death Dis 2010; 1: e18.
121. Wooten MW, Geetha T, Babu JR, et al. Essential role
of sequestosome 1/p62 in regulating accumulation of Lys63-
ubiquitinated proteins. J Biol Chem 2008; 283: 6783–9.
122. Yamamoto A, Tagawa Y, Yoshimori T, et al. Bafilomycin
A1 prevents maturation of autophagic vacuoles by inhibiting fu-
sion between autophagosomes and lysosomes in rat hepatoma
cell line, H-4-II-E cells. Cell Struct Funct 1998; 23: 33–42.
123. Yang Z, Klionsky DJ. Mammalian autophagy: core
molecular machinery and signaling regulation. Curr Opin Cell
Biol 2011; 22: 124–31.
124. Yao KC, Komata T, Kondo Y, et al. Molecular response
of human glioblastoma multiforme cells to ionizing radia-
tion: cell cycle arrest, modulation of the expression of cyclin-
dependent kinase inhibitors, and autophagy. J Neurosurg
2003; 98: 378–84.
125. Ye Y, Shibata Y, Kikkert M, et al. Recruitment of the
p97 ATPase and ubiquitin ligases to the site of retrotransloca-
tion at the endoplasmic reticulum membrane. Proc Natl Acad
Sci USA 2005; 102: 14132–8.
126. Yoon YH, Cho KS, Hwang JJ, et al. Induction
of lysosomal dilatation, arrested autophagy, and cell death
by chloroquine in cultured ARPE-19 cells. Invest Ophthalmol
Vis Sci 2010; 51: 6030–7.
127. Yousefi S, Perozzo R, Schmid I, et al. Calpain-
mediated cleavage of Atg5 switches autophagy to apoptosis.
Nat Cell Biol 2006; 8: 1124–32.
128. Zhang N, Qi Y, Wadham C, et al. FTY720 induces
necrotic cell death and autophagy in ovarian cancer cells: a pro-
tective role of autophagy. Autophagy 2010; 6: 1157–67.
129. Zhu K, Dunner K Jr, McConkey DJ. Proteasome
inhibitors activate autophagy as a cytoprotective response
in human prostate cancer cells. Oncogene 2010; 29: 451–62.
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