Polytechnic University of the philippinesCOLLEGE OF SCIENCE
DEPARTMENT OF BIOLOGY
Chaperon-Mediated Autophagy in Cancer Biology
TERM PAPER
Diana Amor
Jaguio, Nelly MarizPineda, Aljohn
3/10/2012
Chaperon-Mediated Autophagy in Cancer Biology
Cancer
What is cancer?
Cancer is an invasive distensions spreading crab-like as described by
Hippocrates. From the word crab; “karkinos” in Greek and cancer in Latin; came the
name of the disease and the name of its inducing agents, carcinogens. Cellular
biologists had identified cancer with abnormal cell growth in the mid nineteenth century
(Auyang).
Cancer is a potentially fatal disease caused mainly by environmental factors that
mutate genes encoding critical cell-regulatory proteins. The resultant aberrant cell
behavior leads to expansive masses of abnormal cells that destroy surrounding normal
tissue and can spread to vital organs resulting in disseminated disease, commonly a
harbinger of imminent patient death (Alison, 2001; Auyang). The cancerous cells may
occur in liquids, as in leukemia. Mostly, however, occur in solid tumors that originally
appear in various tissues in various parts of the body. By their original locations they
are classified into various types of cancer, such as lung, colon, breast, or prostate
cancer (Auyang).
Although cancer is an ancient disease that afflicts humans and other animals, its
prominence in the Western world rose from the nineteenth century to become “a
disease of civilization (Auyang).” Cancer is primarily a disease of elders; its risk
increases roughly as the fourth power of age (Alison, 2001; Auyang). And it is the
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second-largest common disease—malignant tumors. Coronary disease and cancer
together are responsible for over 80% of all deaths in industrialized countries (Rath,
2001).
The overall 5-year survival rate of many cancers, including liver, lung, pancreas,
bone, and advanced breast cancer, has not increased in the past 30 years. On a
worldwide basis, cancer is fast replacing heart disease as the number one cause of
death in adults (Lam, 2003).
Incidences of cancer keep increasing on a global scale (Rath, 2001). Since 1950,
the overall cancer incidence, particularly in America, has increased by 44%, with breast
cancer and male colon cancer up by 60% and prostate cancer up by 100%. 44% of
Americans living today are expected to develop cancer (Lam, 2003). That is why
Americans have poured roughly $200 billion, in inflation-adjusted dollars, into cancer
research and cancer drug development between 1971 and 2004. Almost one-half of the
bills went to several government agencies, the balance to philanthropies and
pharmaceutical companies (Auyang).
In 1986, the director of National Cancer Institute predicted the eradication of
cancer by 2000. Reality was not anywhere close. In 2004, a new director envisioned
“the elimination of the suffering and death due to cancer by 2015.” The World Health
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Organization estimated that if unchecked, annual global cancer deaths could rise to 15
million by 2020 (Auyang).
What causes cancer?
Cancers evolve through a complicated web of multiple causes and that it is not
only pointless, but also counterproductive, to attempt to assign certain exposures a
certain role in causing cancer (Clapp et. al., 2005).
In 1981, Doll and Peto produced a summary table that estimated that 2% of
cancer deaths were due to pollution and 4% to occupation, with ranges of acceptable
estimates of less than 1% to 5% for the pollution contribution and 2 to 8% for the
occupation contribution. In this same table, they estimate that the proportion of cancer
deaths due to tobacco is 30% and to diet, 35%. A variety of other factors, including
alcohol, food additives, reproduction and sexual behavior, industrial products,
medicines, geophysical factors, and infection are ascribed percentages. The sum of the
individual percentages is 97%, with a final category of “unknown” with no percentage
(Clapp et. al., 2005).
Cancer is a complex genetic disease that is caused primarily by environmental
factors. The cancer-causing agents (carcinogens) can be present in food and water, in
the air, and in chemicals and sunlight that people are exposed to. More significantly, a
globalization of unhealthy lifestyles; particularly cigarette smoking and the adoption of
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many features of the modern world Western diet (Alison, 2001; Auyang). The other
factors include emotional stress, infections, lack of oxygen, poor nutrition, genetic
mutation and environmental pollutants (Alison, 2001; Lam, 2003).
Only about one percent of cancers are unmistakably inherited. Strong genetic
dispositions contribute to a small portion of adult cancers; actually, it contributes
significantly 5-13 percent in cancer incidences (Clapp et. al., 2005; Auyang). Hormone
production during reproductive cycles and other internal factors can also contribute
(Auyang; Lam, 2003).
Inherited genetic defects account for some rare childhood cancers. Variations in
genetic predisposition partly explain why some people are more susceptible than others
are to a particular environmental carcinogen. Many genes involved are not cancer
genes; they do not themselves induce cancer. Rather, they code for enzymes with vital
normal functions, mainly to metabolize chemicals, breaking them down for excretion
(Auyang).
The vast majority of cancers are attributable to what people eat and inhale, how
they behave, their working conditions, viruses and bacteria, and natural and artificial
radiation and chemicals (Clapp et. al., 2005; Auyang); actually, Tobacco use and diet
each account for about 30% of new cancer cases, with infection associated with a
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further 15%; thus, much of cancer is preventable (Alison, 2001). These are usually
called “environmental” risk factors for cancer (Auyang).
However, Rath said in his book, the origin of disease can be considered from two
cellular aspects: The lack of biological fuel needed by the cell’s power plants, the
mitochondria, or a failure in the function of the nucleus, the metabolic control center of
the cell; programming error causes uncontrolled “cell multiplication,” and at the same
time programming error causes a “disruption of the organization of the surrounding
connective tissue,” which enables the diseased cells to spread (2001).
How does it spread in the body?
Cancer does not develop overnight, instead often evolving over many years
(Alison, 2001; Auyang) with detectable premalignant lesions presaging the development
of full-blown malignancy (Alison, 2001).
Cancer cells move through the body with the presence of the cells that are
capable of temporarily dissolving the surrounding tissue—the collagen and elastic fibers
-so it can make its way through. For this purpose the cells use enzymes that can
temporarily digest and weaken the connective fibers surrounding them. All forms of
cancer spread with the help of the tissue-dissolving mechanism. The toxins entering the
body from the diet, such as pesticides and preservatives, are the most common cause
of liver cancer. Also, all pharmaceutical drugs have to be detoxified in the liver.
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Research has established that the more enzymes a cancer cell produces, the more
aggressively the cancer develops. The faster the cancer can spread through a body, the
shorter the life expectancy of the patient if the mechanism is not stopped (Rath, 2001).
The collagen-dissolving mechanism also plays a major role in the spread of
cancer and the growth of secondary tumors in other organs or parts of the body
(metastasis). Small blood vessels provide oxygen and nutrients to tumor cells. The walls
of these blood capillaries are not obstacles for a cancer cell. With the help of collagen-
digesting enzymes, a cancer cell can “eat” its way into the lumen of the small blood
vessel and into the blood stream. The blood can then carry away cancer cells, by which
they can spread and invade other organs (Rath, 2001).
According to Lam, it is the body terrain that determines how the cancer is
expressed. The root of cancer therefore lies in the progress of growth and metastasis,
and not the tissue in which the tumor was first detected (2003).
Metastasis is the process where cancer cells invading the surrounding tissues,
entering the blood stream, spreading and establishing colonies in distant parts of the
body (Auyang). Tumors not only invade surrounding tissue, but are able to colonize
other, often vital, organs, in this process. Widespread metastatic disease is usually a
harbinger of imminent patient death (Alison, 2001).
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What are the kinds of cancer?
Cancer can either ‘benign’ or ‘malignant’. Benign tumours are generally slow-
growing expansive masses that compress rather than invade surrounding tissue. As
such they generally pose little threat, except when growing in a confined space like the
skull, and can usually be readily excised. However, many so-called benign tumours
have malignant potential, notably those occurring in the large intestine, and these
should be removed before malignancy develops. Malignant tumors are usually rapidly
growing, invading surrounding tissue and, most significantly, colonizing distant organs.
The ability of tumour cells to detach from the original mass (the primary tumour) and set
up a metastasis (secondary tumour) discontinuous with the primary is unequivocal proof
of malignancy. Tumours are also classified according to their tissue of origin;
recognition of the parent tissue in a lymph node metastasis could establish the location
of a hitherto undiagnosed primary tumour (Alison, 2001).
Cancers that are not inherited are called “sporadic.” This means not that they
have no genetic component but that their genetics occurs not in germ cells but in
somatic cells, which constitute the bulk of our body. Some somatic cells, such as
muscle cells or neurons in the brain, stop dividing upon maturity. They can grow bigger
in size or establish more connections, but their numbers do not multiply. Cancer
seldom if ever appears in such non-dividing cells. It appears in tissues where cells die
and are replenished by new cell divisions (Auyang).
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What are the cures for cancer?
According to Lam in 2003, there is no “single cure” for cancer and none is
proposed. For the past 50 years, war on cancer has been fought with three tools –
surgery (cut), radiation therapy (burn), and chemotherapy (poison). Localized tumors
can be removed by surgery or irradiation with high survival rates (Auyang). Benign
tumours can normally be removed by surgery. Malignant solid tumors will, if possible, be
surgically resected, probably followed and even preceded by other treatment modalities.
If the tumour is amenable to surgery, then surgery is the single most effective tool in the
anticancer armamentarium. Targeted radiotherapy is another option, as are
combinations of anticancer drugs (Alison, 2001).
Most conventional anticancer drugs have been designed with deoxyribonucleic
acid (DNA) synthesis as their target. Therein lies the problem, in that tumor cells are not
the only proliferating cells in the body; cells that line the alimentary tract, bone marrow
cells that generate red blood cells and cells to fight infection, and epidermal cells
including those that generate hair are all highly proliferative. Thus, patients with cancer
receiving chemotherapy commonly su er unwanted (hair loss) and sometimesff
potentially life-threatening side e ects that limit treatment (Alison, 2001). ff
At a cellular level cancer is a very rare disease given that an individual has many
millions of cells, so normally the repair and/or elimination mechanisms of damaged cells
must be very e cient, asking to have a ‘caretaker’ function. To account for the multipleffi
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mutations in cancer cells, attention has become focused on the mechanisms of DNA
metabolism that maintain genome integrity, looking for the so-called ‘mutator
phenotype’. If the mechanisms of DNA repair are faulty, this leads to ‘genetic instability’,
facilitating an increased rate of alterations in the genome (Alison, 2001).
However, naturally oriented physicians think of the body as a closed internal
ecosystem, and believe that it is the dysfunction of this ecosystem that is primarily
responsible for the development of cancer. According to him, no treatment,
conventional or otherwise, can completely eliminate all cancer cells. The reason is
simple. Cancer is a systemic disease, and there are simply too many cancerous or pro-
cancerous cells within the ecosystem of the body. Cancer is not a localized problem but
a whole-body phenomenon of metastatic growth. Its growth process is affected by
biological conditions. He then, therefore proposed fights against cancer by optimizing
the internal terrain and enabling the patient’s internal system to destroy the tumor. It
enhances the patient’s health so that cancer cells cannot grow and multiply (Lam,
2003).
Rath proposed a way of preventing the spread of the cancer cells. He said that
the nature itself provides us with two large groups of molecules that can block collagen
digestion and its dissolving actions which lead to the spread of cancer. The first group is
the body’s intrinsic enzymatic block that can stop the action of collagen-digesting
enzymes in a few moments. The second group is the enzyme- blocking substances that
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come from our diet or as dietary supplement. The most important one in this group is
the natural amino acid L-lysine. When lysine is supplied in a sufficient amount as a
dietary supplement; it can block the anchor sites in the connective tissue that collagen-
digesting enzymes use to attach themselves to the tissue. In this way lysine prevents
these enzymes from uncontrollably disintegrating connective tissue (2001).
Lam said that, the use of non-toxic natural therapies has achieved huge
successes over the past few decades. Extensive studies have proven them to have an
edge over conventional therapies success rate of natural treatment is so much better
than for many conventional cancer treatments (2003).
More than cures, scientists are cautiously optimistic about the possibility of
improving early detection and prevention of cancer. Cancer takes several steps and a
long time to develop. Its long latent period gives many opportunities to catch cells in
their early stages of mutation and intervene to stop cancer progression. For instance,
the pap smear followed by surgical removal of detected lesions have reduced death rate
of cervical cancer by almost 80 percent. To extend the success in cervical cancer to
cancer in general, scientists strive to identify biological markers that can finger incipient
cancerous cells and predict whether they will evolve to significant cancer (Auyang).
Alison said that, no individual can guarantee not to contract the disease, but it is
so strongly linked to diet and lifestyle that there are plenty of positive steps that can be
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taken to reduce the chances: eat more fruit and vegetables, reduce the intake of red
meat and definitely do not smoke. Carcinogens interact with the individual’s constitution,
both inherited and acquired, determining vulnerability to cancer induction (2001).
Chaperone-Mediated Autophagy
Chaperone-mediated autophagy (CMA) is an intracellular catabolic pathway that
mediates the degradation of a selective subset of cytosolic proteins in lysosomes.
Autophagy or self-eating is broadly used to designate the lysosomal delivery and
degradation of intracellular components (Yang and Klionsky, 2010). Various types of
autophagy co-exist in almost all cells, and they can be differentiated by the mechanisms
that mediate the delivery of cargo to lysosomes. Macroautophagy and microautophagy
are kinds of the autophagic process, in which entire regions of cytosol or selective
cytosolic components are sequestered in vesicular compartments. Lysosomal enzymes
can gain access to the enclosed cargo through direct fusion of the vesicles with
lysosome, or by internalization of cargo-containing vesicles that form at the lysosomal
membrane. A third form of autophagy, solely dedicated to degradation of soluble
proteins can also be detected in most cell types in mammals. This autophagic process,
known as chaperone-mediated autophagy, differs from the other forms of autophagy in
both the way in which cargo proteins are recognized for lysosomal delivery and the way
in which these proteins reach the lysosomal lumen (Dice, 2007; Cuervo, 2010).
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CMA is a process activated during long term starvation in which cells selectively
degrade proteins in order to recycle their amino acids or use them for energy. During
nutrient deprivation, substrates that contain a consensus motif related to KFERQ are
recognized by a chaperone-cochaperone complex containing the heat shock cognate
protein of 70 kDa (hsc70). Once this chaperone-cochaperone complex binds the
substrate, it docks on the lysosomal membrane via a receptor known as the lysosomal
associated membrane protein 2a or lamp2a. The substrate then is unfolded,
presumably by the chaperone-cochaperone complex, translocated into the lumen with
the help of a lysosomal isoform of hsc70, and degraded. Like most organelle protein
import pathways, CMA is saturable as well as temperature-dependent. The substrates
for CMA also compete with one another for binding and import, which provides an
experimental method for discovering new substrates. There have been several
substrates identified for CMA including ribonuclease A and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH).
The cytosolic chaperones and co-chaperones that participate in CMA are also
involved in other intracellular pathways this was according to Chiang et al., 1989.
According to Cuervoet al., (1997), the chaperones located in the lysosomal lumen or
associated to the lysosomal membrane – namely lys-Hsc70, membrane associated
Hsc70 and lys-Hsp90– appear to be exclusively dedicated to CMA. However, as these
are post-translational variations of cytosolic chaperones rather than independent gene
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products, regulating their expression does not provide a mean for selectively affecting
CMA activity.
Moreover, Cuervo and Dice (1996) determined that LAMP-2A, the membrane
protein that acts as a receptor for the CMA substrates has often been manipulated to
regulate CMA activity. In fact, levels of LAMP-2A at the lysosomal membrane directly
determine rates of CMA activity, because substrate binding to the cytosolic tail of
LAMP-2A is a limiting step in CMA. Eskelinenet al., (2005) added that LAMP-2A is a
spliced variant of a single Lamp2 gene, which also encodes two other variants, LAMP-
2B and LAMP-2C with identical luminal regions but different transmembrane and
cytosolic tails. Substrate binding to the LAMP-2A cytosolic tail does not occur at the
KFERQ-targeting region and a designated LAMP-2A-binding motif in the substrate has
not been identified yet. However, the fact that substrate binding requires the four
positive charges in the LAMP-2A cytosolic tail suggests that electrostatic interactions,
rather than specific amino acid residues, mediate substrate binding.
The function of LAMP-2A extends beyond that of a receptor as this protein is also
an essential component of the CMA translocation complex (Bandyopadhyay et al.,
2008). Binding of substrate proteins to LAMP-2A monomers drives its organization into
a 700 kDamultimeric complex at the lysosomal membrane. The motif present in the
transmembrane region of LAMP-2A is important for multimerization. We have shown
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that mutations that prevent multimerization abolish substrate translocation but not
substrate binding to LAMP-2A (Bandyopadhyay et al., 2008).
Although, in contrast to other translocation systems, luminal chaperones do not
form part of a stable translocon unit in CMA, they are still essential for substrate uptake.
In fact, a form of hsc70 resident in the lysosomal lumen is needed for complete
translocation of substrate proteins into lysosomes. Incubation of cultured fibroblasts with
blocking antibodies against hsc70 that reach the lysosomal lumen through endocytosis
exerts a strong inhibitor effect on CMA. Furthermore, levels of lys-hsc70 have helped
identify subgroups of lysosomes that manifest different ability to perform CMA. Only
those lysosomes containing hsc70 in their lumen are competent for uptake of CMA
substrates. Interestingly, the percentage of hsc70-containing lysosomes, which is no
more than 40% under resting conditions, escalates to 80% in liver under conditions in
which CMA is up-regulated, such as during prolonged starvation or mild oxidative
stress. This increase in the amount of lysosomes competent for CMA is a consequence,
at least in part, of changes in the luminal acidification in these organelles. Thus, lys-
hsc70 is stable in the lysosomal lumen at a pH of around 5.2, but a slight increase in the
lysosomal pH is enough to destabilize this protein and make it amenable to degradation
by the abundant lysosomal protease. Many factors could contribute to transient changes
in lysosomal pH and the subsequent destabilization of hsc70. Among them, we have
recently identified that fusion of lysosomes with autophagosomes when
macroautophagy is maximally activated contributes to a dissipation of pH enough to
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render hsc70 unstable, and results in decreased CMA activity. The pH dependence of
lys-hsc70 may thus constitute a novel regulatory node in the cross-talk between
macroautophagy and CMA.
The study of CMA in particular cell types and the characterization of the
degradation of specific cellular proteins by CMA are behind the cell type–specific
functions recently proposed for this pathway. CMA activity has been linked to the
regulation of cellular proliferation in tubular kidney cells through the degradation of Pax-
2. Levels of this transcription factor, an essential regulator of kidney cell proliferation
and differentiation, are controlled through its degradation by CMA. Consequently,
changes in CMA activity may modulate kidney organogenesis and growth. Similarly, a
role for CMA in antigen presentation has been proposed in dendritic cells.
Macroautophagy has been shown to contribute to both the presentation of
endogenous peptides on major histocompatibility complex class II molecules, as well as
the presentation mediated by MHC class I molecules. Although relatively limited
information is available on the contribution of CMA to immunity, recent studies have
shown that reduction of LAMP-2A or hsc70 levels decreases presentation via MHC
class II. Interestingly, pharmacological inhibition of hsp90, which will reduce CMA
activity, also resulted in decreased antigen presentation in a second independent study.
Antigen processing and loading usually occurs in endosomes rather than in secondary
lysosomes, as the lower proteolytic capacity of the former compartment allows
preservation of the presenting peptides and their loading on MHC class II molecules.
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However, to date, CMA has only been described to take place in secondary lysosomes.
Future studies are required to determine whether this hsc70-mediated presentation of
antigens takes place in late endosomes or in lysosomes.
Lysosomal storage disorders (LSDs) are a group of genetic diseases resulting
from loss of a specific lysosomal enzyme activity, and the consequent accumulation of
its substrates in the lysosomal compartment. The enzymatic impairment can result from
the malfunctioning of a specific enzyme in lysosomes, or from failure in its delivery to
this compartment. CMA, like any other type of autophagy, is likely to be indirectly
affected in these pathologies, because lysosomes are the final compartment for all
autophagic pathways. However, a direct connection to CMA has been recently
established with two different LSDs which includesgalactosialidosis and mucolipidosis
type IV. Patients with galactosialidosis lack cathepsin A, a protein that acts as
chaperone for different lysosomal enzymes, but that has also been recently shown to
participate in LAMP-2A turnover. The inability to properly degrade LAMP-2A in the cells
from these patients results in abnormally high rates of CMA. In the case of patients with
mucolipidosis type IV, who bear a mutation in the transient receptor potential mucolipin-
1, CMA activity decreases. The fact that hsc70 interacts with this receptor has led to the
proposition that altered docking of hsc70 at the lysosomal membrane could be behind
the observed decrease in CMA. However, another possible explanation that requires
further testing is that the small molecule channeling activity of this membrane protein is
required for CMA.
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Although the classification as LSD has been traditionally restricted to defects in
enzymatic activity, recently, alterations in nonenzymaticlysosomal proteins, such as
membrane proteins, have also been shown to result in lysosome malfunctioning and
substrate accumulation. Among these new forms of LSD, of particular relevance for
CMA is Danon disease, a vacuolar myopathy that originates from a primary defect in
the lamp2 gene. Although, as indicated in previous sections, elimination of the three
protein variants of this gene will result in a complex phenotype, it is anticipated that
patients with Danon disease will also have reduced CMA activity.
Chaperone-Mediated Autophagy in Cancer Biology
Autophagy plays a crucial role in maintaining neuronal homeostasis through
clearance of defective organelles and unfolded/aggregating proteins. Knockout of
autophagy pathway genes leads to accumulation of poly-ubiquitinated protein
aggregates and can result in neurodegeneration, and motor and behavioral deficits in
mice. Also, autophagy interacts with other protein degradationand vesicular trafficking
pathways. While autophagy can at least partially substitute for reduced proteasomal
activity and vice versa, the disturbance of the endosomal-lysosomal system disrupts
autophagy and reduced autophagy impairs endosomal-lysosomal trafficking. It clears
neurotoxic proteins. The activation of this reduces the toxicity of aggregation prone
proteins, while inhibition of autophagy impairs their clearance and causes enhanced
cellular stress and neurodegeneration. This can also be a cellular death pathway, which
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isactivated in neurons after acute injury and inhibition of autophagy under those
conditions can reduce neurodegeneration. However, autophagy is impaired in the final
stages of most neurodegenerative diseases.
Numerous observations suggest the existence of strong links between autophagy
and cancer. Several tumor suppressor genes stimulate autophagy, whereas oncogenes
are known that inhibit autophagy. The connection between both processes is probably
related to the overlap that exists between the pathways involved in regulation of
autophagy and tumorigenesis. Mutations that affect the function of mTOR or Beclin 1
have been identified in human cancers (Cao, 2007).The function of mTOR overlaps
with signaling pathways involved in tumorgenesis. Several tumor suppressor genes
have been shown being involved in the upstream inhibition of mTOR signaling and in
this way stimulate autophagy. Moreover, oncogene proteins are known that activate
mTOR. The importance of Beclin 1 in human cancers is illustrated by the fact that
mono-allelic deletions in Beclin 1 occur in a 40 – 75 % of cases of human breast,
ovarian and prostate cancer. Whether this only relates to the function of Beclin 1 in
autophagy or also to autophagy-independent functions of Beclin1 is not yet known
(Cecconi and Levine, 2008).
Selectively inhibited CMA by knocking down the LAMP-2A lysosomal receptor in
cultured cancer cell lines and in mice carrying human primary lung tumor xenograft s
using short hairpin RNAs (shRNAs). Although such an approach is the best available
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method to impair CMA in tumor cells, it does raise an important cave at as it is formally
possible that LAMP-2A may possess additional functions that are separate from its role
in CMA. Nonetheless, even bearing this in mind, the results here are provocative
because they demonstrate that impaired CMA is sufficient to reduce tumor cell
proliferation rates both in vitro and in vivo. These effects are CMA-specific because
knockdown of a gene that is essential for macroautophagy (atg7) had little effect on
tumor growth. Inhibition of the CMA pathway also reduces the metastatic potential of the
tumor cells, which is extremely important from a clinical perspective given that
metastatic disease remains the principal cause of cancer mortality. In the real world,
most treatment is targeted to patients presenting with established cancers (Kon et al.,
2011) to ask whether an established tumor is affected when CMA is blocked by viral
delivery of shRNAs that target LAMP-2A. They found that the direct injection of
lentivirus encoding LAMP-2A shRNAs into tumor xenogra s caused marked regression
that was associated with increased tumor cell death as well as reduced staining for Ki-
67, which is a standard marker for tumor cell proliferation.
Thus, it appears that CMA is required for optimal tumor growth and metastasis to
distant sites and that targeting CMA in established tumors can induce the tumor cells to
slow their growth and undergo apoptosis leading to tumor regression. How does
blockade of the CMA pathway elicit such profound antitumor phenotypes. At least part
of the explanation is related to changes in cellular metabolism in CMA-defi cient cancer
cells. Bioenergetic assays indicated that inhibiting CMA produced a decrease in the
glucosedependent extracellular acidifi cation rate (ECAR), a finding consistent with
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reduced glycolysis. In contrast, no changes in oxygen consumption or oxidative
metabolism were eadily evident in the CMA-deficient cancer cells. Increased aerobic
glycolysis is a well-known characteristic of tumor cells, commonly termed the “Warburg
effect”; moreover, abundant data indicate that reduced glycolytic metabolism, such as
observed here are CMA blockade, can profoundly attenuate both energy (adenosine
triphosphate) production and biosynthetic capacity, which are vital for cancer cell growth
and proliferation. Notably, this reduction in glycolysis in CMA-decient cancer cells was
associated with a decrease in several glycolytic enzymes. This result is somewhat
counterintuitive because these glycolytic enzymes possess KFERQ motifs and are
known substrates for CMA-mediated degradation; as a result, one would expect that
blocking CMA should result in an increase in these glycolytic enzymes, rather than the
decrease observed. The authors propose that this decrease might be at least partly due
to activation of the tumor suppressor p53 in CMA-defi cient cells, which results in the
transcriptional downregulation of multiple glycolytic enzymes. Nonetheless, because
CMA activity is upregulated in cancer cells independent of their p53 status, it is very
likely that CMA deficiency inhibits tumorigenesis by other mechanisms in addition to
p53-mediated suppression of glycolysis.
One of the main mechanisms by which macroautophagy potentially suppresses
tumor development is by eliminating stress-related molecules, such as p62/SQSTM1,
as well as oxidized, damaged proteins (Matthew, etal. 2009 ). This does not seem to
apply to CMA because there were no apparent differences in p62/SQSTM1 protein
levels and no increases in oxidized or aggregated proteins when the CMA pathway was
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inhibited. Notably, the authors demonstrate that CMAdeficient cancer cells display a
compensatory increase in proteasomal degradation of CMA protein substrates, which
appears to be critical for maintaining protein quality control and mitigating the effects of
oxidative stress. Second, are the mechanisms underlying the antitumor effects of CMA
inhibition similar to those underlying the effects of macroautophagy inhibition. Despite
the compensatory increase in proteasomal degradation, an intriguing possibility is that
there is accumulation of specific target proteins containing the KFERQ motif, resulting in
the phenotypic changes observed in cells with a defective CMA pathway. In principle,
approximately one-third of the proteome can be targeted for CMA-mediated degradation
because about 30% of proteins contain the hsc70-targeting sequence (Arias and
Cuervo, 2011). However, the number of definitively identified CMA substrates is much
smaller than this. Hence, if currently unknown CMA substrates do indeed exist in cancer
cells, identifying these molecules will be key to understanding the precise mechanisms
through which CMA is mediating tumor development.
Most important of all, how do these findings relate to better treatments for cancer,
The data presented in the Kon et al. paper suggest that CMA inhibitors could be useful
for cancer therapy, as they should inhibit tumor growth and also reduce the ability of
tumor cells to metastasize. From a therapeutic standpoint, an important limitation is that
we do not currently have a feasible method to selectively inhibit CMA in patients (unlike
in the mouse xenograft model). Thus, we are unable to reliably reduce LAMP-2A protein
concentrations within tumor cells in people and, to date, we lack pharmacological
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inhibitors that selectively block the CMA pathway. We do, however, have drugs that
more broadly inhibit all forms of lysosomal degradation. The most widely used of these
include chloroquine and hydroxychloroquine, which are being tested in a number of
clinical trials (more than 30 such trials are currently listed on the ClinicalTrials.gov Web
site) in combination with other anticancer drugs (Aramavadi, etal., 2011). The rationale
behind these trials is to block macroautophagy, but the question is whether CMA will
also be inhibited in these patients. Therefore, should there be any beneficial results
from these trials, we would need to consider whether it is CMA rather than
macroautophagy that is the critical target of chloroquine or hydroxychloroquine. Last,
but not least, like all anticancer strategies, it seems unlikely that all tumors will respond
equally well to CMA inhibition. Therefore, it will be critical to identify markers that
predict the dependence of tumors on CMA before blocking this pathway. In this regard,
the interconnections between macroautophagy and CMA will be important to uncover.
Remarkably, when tumor cells are transformed due to mutation of the small GTPase
Ras, this leads to a requirement for macroautophagy that has been proposed to
facilitate glycolysis (Lock, etal., 2011). Similarly, it has been suggested that the
presence of oncogenic Ras mutations makes tumor cells “addicted” to
(macro)autophagy (Guo, 2011). Accordingly, tumor types in which Ras mutations are
particularly common, such as pancreatic cancer, have been found to be particularly
sensitive to growth inhibition by chloroquine (Yang, 2011). These studies point to
specific patient subsets, such as those with tumors harboring Ras mutations, where
inhibition of macroautophagy may be particularly useful. Interestingly, several of the
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lung cancer cell lines used by Kon et al. possess K-Ras mutations, immediately raising
the question of whether Ras-transformed cells are similarly addicted to CMA and
whether CMA inhibition, rather than macroautophagy inhibition, dictates the response to
chloroquine observed in the aforementioned studies. The answers to these and the
many other questions that arise from the exciting new study of Kon et al. will no doubt
keep cancer investigators working on autophagy very busy. However, what seems clear
is that it is unwise for cancer biologists to exclusively focus on macroautophagy in tumor
cells at the expense of CMA. If we continue to do so, we are certain to overlook some of
the most important aspects of autophagy in cancer biology.
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