U.S. patent application number 13/895497 was filed with the patent office on 2014-05-15 for therapeutic modulation of autophagy.
This patent application is currently assigned to Rutgers, The State University of New Jersey. The applicant listed for this patent is Cristina Karp, Robin Mathew, Anne Marie Storhecker, Eileen White. Invention is credited to Cristina Karp, Robin Mathew, Anne Marie Storhecker, Eileen White.
Application Number | 20140134661 13/895497 |
Document ID | / |
Family ID | 42731031 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140134661 |
Kind Code |
A1 |
White; Eileen ; et
al. |
May 15, 2014 |
THERAPEUTIC MODULATION OF AUTOPHAGY
Abstract
Methods for screening for modulators of autophagy are disclosed.
Methods for identifying genes whose expression inhibits autophagy,
as well as genes whose expression promotes autophagy, are
disclosed. Also disclosed are methods for identifying compounds
that stimulate autophagy, as well as compounds that inhibit
autophagy. Cell lines that may be used in the methods of
identification are also disclosed.
Inventors: |
White; Eileen; (Princeton,
NJ) ; Storhecker; Anne Marie; (New York, NY) ;
Mathew; Robin; (Monroe, NJ) ; Karp; Cristina;
(North Brunswick, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
White; Eileen
Storhecker; Anne Marie
Mathew; Robin
Karp; Cristina |
Princeton
New York
Monroe
North Brunswick |
NJ
NY
NJ
NJ |
US
US
US
US |
|
|
Assignee: |
Rutgers, The State University of
New Jersey
New Brunswick
NJ
|
Family ID: |
42731031 |
Appl. No.: |
13/895497 |
Filed: |
May 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13440596 |
Apr 5, 2012 |
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13895497 |
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12622410 |
Nov 19, 2009 |
8187802 |
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13440596 |
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61116085 |
Nov 19, 2008 |
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Current U.S.
Class: |
435/29 |
Current CPC
Class: |
G01N 33/5008 20130101;
G01N 33/53 20130101; G01N 33/5023 20130101; C12Q 1/6809 20130101;
C12Q 1/025 20130101 |
Class at
Publication: |
435/29 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The present application was supported in part by the
National Institutes of Health under Grant Nos. R37 CA53370 and RO1
CA130893 and the Department of Defense under DOD W81XWH06-1-0514
and DOD W81XWH05. The government may have certain rights in the
invention.
Claims
1.-13. (canceled)
14. A method of identifying; a gene whose expression modulates
autophagy comprising: (A) infecting a test cell expressing a marker
of protein aggregation with shRNA from a lentivirus library,
wherein lentiviral infection introduces an shRNA directed to a
target gene into the test cell and wherein expression of the shRNA
in the test cell causes lowered expression of the target gene: (B)
subjecting the infected test cell to metabolic stress and recovery
therefrom; (C) performing analysis on the infected test cell to
determine the level of protein aggregates comprising the marker;
and (D) comparing the level of protein aggregates comprising the
marker in the test cell infected with shRNA to a target gene with
the level protein aggregates comprising the marker in a cell
infected with control shRNA: wherein a higher or lower level of
aggregates comprising the marker in the test cell infected with an
shRNA to a target gene compared to the level of aggregates observed
in the cell infected with the control shRNA indicates that the
target gene encodes an inducer or inhibitor of autophagy.
15. The method of claim 14 wherein the marker comprises
p62/Sequestosome 1 protein linked to a label molecule.
16. The method of claim 14 wherein the test cell is
autophagy-defective.
17. The method of claim 16 wherein the autophagy-defective cell
haploinsufficient for an autophagy gene.
18. The method of claim 16 wherein the autophagy-defective cell is
mill for an autophagy gene.
19. The method of claim 14 wherein the test cell is
apoptosis-defective.
20. The method of claim 14 wherein the test cell is an immortalized
baby mouse kidney cell.
21. The method of claim 14 further comprising the step of
validating lowered expression of the target gene following shRNA
infection to ensure knockdown:phenotype correlation of the
candidate autophagy modulator.
22. A method for identifying an inducer or inhibitor of autophagy
comprising the steps of: (A) contacting a test cell expressing a
marker of protein aggregation with a compound; (B) subjecting the
test cell to metabolic stress and recovery therefrom; (C)
performing analysis on the test cell to determine the level of
protein aggregates comprising the marker; and (D) comparing the
level of protein aggregates comprising the marker in the test cell
with that of a control cell not contacted with the compound,
wherein either a higher or lower level of aggregates comprising the
marker in the test cell indicates that the compound is capable of
modulating autophagy.
23. The method of claim 22 wherein the marker comprises
p62/Sequestosome 1 protein linked to a label molecule.
24. The method of claim 22 wherein the test cell is
autophagy-defective.
25. Them method of claim 24 wherein the autophagy-defective cell is
haploinsufficient for an autophagy gene.
26. The method of claim 24 wherein the autophagy-defective cell is
mill for an autophagy gene.
27. The method of claim 22 wherein the test cell is
apoptosis-defective.
28. The method of claim 22 wherein the test cell is an immortalized
baby mouse kidney cell.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of U.S. Provisional
Application No. 61/116,085, filed Nov. 19, 2008, the disclosure of
which is hereby incorporated by reference herein, in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Macroautophagy (autophagy) is an important mechanism for
targeting cellular components including proteins, protein
aggregates and organelles for degradation in lysosomes. This
catabolic, cellular self-digestion process is induced in response
to starvation or stress, causing the formation of double membrane
vesicles called autophagosomes that engulf proteins and organelles.
Autophagosomes then fuse with lysosomes where the autophagosome and
their cargo are degraded. This lysosome-mediated cellular
self-digestion serves to recycle intracellular nutrients to sustain
cell metabolism during starvation and to eliminate damaged proteins
and organelles that accumulate during stress. Although elimination
of individual proteins occurs by the ubiquitin-mediated proteasome
degradation pathway, the autophagy pathway can eliminate protein
aggregates and organelles. Thus, autophagy complements and overlaps
with proteasome function to prevent the accumulation of damaged
cellular components during starvation and stress. Through these
functions, autophagy is an essential cellular stress response that
maintains protein and organelle quality control, protects the
genome from damage, and sustains cell and mammalian viability.
[0004] Autophagy is thought to be controlled by ATG proteins,
initially identified in yeast, for which there are mammalian
homologues (Levine, B., and Kroemer, G. (2008), Autophagy in the
pathogenesis of disease, Cell 132, 27-42). ATG proteins are
comprised of kinases, proteases, and two ubiquitin-like conjugation
systems that likely function in concert with a host of unknown
cellular proteins to control autophagosome formation, cargo
recognition, engulfment, and trafficking to lysosomes.
[0005] In mice, autophagy enables survival of neonatal starvation
by preventing energy depletion. Mice with targeted
autophagy-deficiency (atg5.sup.-/- or atg7.sup.-/-) in the brain
accumulate damaged mitochondria and polyubiquitin-containing
protein aggregates, and display neuronal degeneration. Defects in
autophagy through liver-specific atg7 deletion in mice similarly
results in protein aggregate accumulation, hepatocyte cell death
and severe liver injury. These findings support a prosurvival role
for autophagy in sustaining cellular metabolism and maintaining
protein and organelle quality control by eliminating damaged
proteins and organelles that are particularly important during
nutrient stress and aging (Levine, B. and Kroemer, G., (2008)).
[0006] Autophagy dysfunction is a major contributor to diseases
including, but not limited to, neurodegeneration, liver disease,
and cancer. Many human neurodegenerative diseases are associated
with aberrant mutant and/or polyubiquitinated protein accumulation
and excessive neuronal cell death. Neurons of mice with targeted
autophagy defects accumulate polyubiquitinated- and p62containing
protein aggregates that result in neurodegeneration. The human
liver disease steatohepatitis and a major subset of hepatocellular
carcinomas (HCCs) are associated with the formation of
p62-containing protein aggregates (Mallory bodies) (Zatloukal, K.,
et al. (2002), p62 is a common component of cytoplasmic inclusions
in protein aggregation diseases, Am. J. Pathol. 160, 255-263).
Livers of mice with autophagy defects have p62-containing protein
aggregates, excessive cell death, and HCC.
[0007] Autophagy is also induced by stress and starvation in tumor
cells where it predominantly provides a prosurvival function.
Metabolic stress is common and autophagy localizes to
metabolically-stressed tumor regions. Autophagy has been identified
as an important survival pathway in epithelial tumor cells that
enables long-term survival to metabolic stress (Degenhardt, K., et
al. (2006), Autophagy promotes tumor cell survival and restricts
necrosis, inflammation, and tumorigenesis, Cancer Cell 10, 51-64;
Jin, S., and White, E. (2007), Role of autophagy in cancer:
management of metabolic stress. Autophagy 3, 28-31;
Karantza-Wadsworth, V., et al., (2007), Autophagy mitigates
metabolic stress and genome damage in mammary tumorigenesis, Genes
Dev 21, 1621-1635; Mathew, R. et al., (2007a), Role of autophagy in
cancer, Nat Rev Cancer 7, 961-967; Mathew, R., et al. (2007b),
Autophagy suppresses tumor progression by limiting chromosomal
instability, Genes Dev 21, 1367-1381). Tumor cells with defined
defects in autophagy accumulate p62-containing protein aggregates,
DNA damage, and die in response to stress, whereas those with
intact autophagy can survive for weeks utilizing the autophagy
survival pathway. Thus, autophagy may be required to prevent tumor
cell damage and to maintain metabolism. Tumor cells can exploit
this survival function to remain dormant only to reemerge under
more favorable conditions.
[0008] Paradoxically, autophagy defects through allelic loss of the
essential autophagy gene beclin1 or through constitutive activation
of the autophagy-suppressing PI-3 kinase/mTOR pathway are common in
human tumors. Roughly half of human cancers may have impaired
autophagy, either due to constitutive activation of the PI-3 kinase
pathway or allelic loss of the essential autophagy gene beclin1,
rendering them particularly susceptible to metabolic stress and
autophagy inhibition (Jin et al., 2007; Jin, S., and White, E.
(2008), Tumor suppression by autophagy through the management of
metabolic stress, Autophagy 4, 563-566).
[0009] Analogous to a wound-healing response, chronic tumor cell
death in response to stress and induction of inflammation and
cytokine production may provide a non-cell-autonomous mechanism by
which tumorigenesis is promoted in autophagy-defective cells.
Autophagy-defective tumor cells also display an elevated DNA damage
response, gene amplification and chromosome instability in response
to stress, suggesting that autophagy limits genome damage as a
cell-autonomous mechanism of tumor suppression. Possible
non-mutually exclusive mechanisms by which autophagy may protect
the genome include maintenance of metabolism and ATP levels,
reduction of oxidative stress, and elimination of damaged protein
and organelles.
[0010] The importance of autophagy in cellular garbage disposal is
clear, as autophagy is the only identified mechanism for the
turnover of large cellular structures such as organelles and
protein aggregates. How organelles are recognized and directed to
autophagosomes for degradation may involve organelle-specific
processes such as mitophagy and ER-phagy that may mitigate
oxidative stress emanating from dysfunctional organelles. Damaged
proteins that accumulate during stress can be refolded,
ubiquitinated and degraded by the proteasome pathway, or aggregated
and degraded by autophagy. To direct damaged or unfolded proteins
to the autophagy pathway, p62 binds to polyubiquitinated proteins
forming protein aggregates by oligomerization and to Atg8/LC3 on
the autophagosome membrane to target aggregates to autophagosomes
for degradation. Protein aggregation may be a protective mechanism
to limit cellular exposure to toxic proteins through sequestration,
as well as an efficient packaging and delivery mechanism that
collects and directs damaged proteins to autophagosomes.
Liver-specific autophagy defects in mice cause accumulation of p62
aggregates, elevated oxidative stress and hepatocyte cell death.
Thus, without seeking to be bound by any theory or theories of
operation, it is believed that the inability to dispose of p62
aggregates through autophagy may be toxic to normal tissues.
[0011] The ATG6/Beclin1-Vps34-ATG8/LC3 complex regulates
autophagosome formation; LC3 cleavage, lipidation, and membrane
translocation are frequently utilized to monitor autophagy
induction. The mechanism by which starvation and stress activate
autophagy is controlled in part through the PI-3 kinase pathway via
the protein kinase mTOR. Growth factor and nutrient availability
promote mTOR activation that suppresses autophagy, whereas
starvation and mTOR inactivation stimulate autophagy (Klionsky
(2007), Nat Rev Mol Cell Biol 8, 931-937). While there are other
mechanisms to regulate autophagy, mTOR provides a link between
nutrient and growth factor availability, growth control, autophagy,
and metabolism.
[0012] Autophagy is believed to play an essential role in
maintaining protein quality control, while defective autophagy may
be involved in the development of diseases including, but not
limited to, neurodegeneration, steatohepatitis, and cancer.
Therefore, there exists a need for identification of stimulators of
autophagy.
[0013] Additionally, there exists a need for the identification of
inhibitors of the autophagy survival pathway in, for example,
cancer cells. Such inhibitors of autophagy could be used in the
prevention and/or treatment of cancer.
BRIEF SUMMARY OF THE INVENTION
[0014] In certain aspects, the present invention relates to methods
of identifying modulators of autophagy. Certain aspects relate to
methods of identifying inhibitors of autophagy. Other aspects
relate to methods of identifying stimulators of autophagy.
Additional aspects relate to methods of identifying genes whose
expression affects autophagy.
[0015] One aspect of the invention relates to a method comprising
the steps of: A) screening an shRNA library using a test cell that
expresses an autophagosome marker; (B) subjecting the cell to
metabolic stress; and (C) performing analysis on the cell to
determine the localization of the autophagosome marker in the test
cell.
[0016] Another aspect of the present invention relates to a method
for identifying inhibitors of autophagy comprising the steps of:
(A) contacting a test cell that expresses an autophagosome marker
with a compound; (B) subjecting the test cell to metabolic stress;
and (C) performing analysis on the test cell to determine the
localization of the autophagosome marker in the cell.
[0017] In one aspect, the present invention relates to a method
comprising: (A) screening an shRNA library using an
autophagy-defective test cell expressing a marker of protein
aggregation; (B) subjecting the test cell to metabolic stress; and
(C) performing analysis on the test cell to determine the level of
the marker.
[0018] In another aspect, the present invention relates to a method
for identifying stimulators of autophagy comprising the steps of:
(A) contacting a test cell expressing a marker of protein
aggregation with a compound; (B) subjecting the cell to metabolic
stress; and (C) performing analysis on the test cell to determine
the level of the marker.
[0019] Additional aspects relate to cells and cell lines that may
be used in certain embodiments of the invention.
[0020] Both the foregoing general description and the following
detailed description are exemplary and explanatory, and are
intended to provide further explanation of the invention as
claimed. Other aspects, advantages, and novel features will be
readily apparent to those skilled in the art from the following
detailed description of the invention.
[0021] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 illustrates elevated and persistent p62 in
autophagy-defective tumor cells under metabolic stress. (A) Domain
organization of p62 (SQSTM1/Sequestosome 1) illustrating the Phox
and Bem1p (PB1) domain that assists in oligomerization and binding
to atypical Protein Kinase C (aPKC), the Zinc finger (ZZ) domain
that binds to the receptor interacting protein (RIP), the TRAF6
binding site (TBS) involved in NF-.kappa.B signaling, the
microtubule associated protein light chain 3 (LC3) binding domain
that interacts with LC3/ATG8, and the ubiquitin-associated (UBA)
domain that binds poly-ubiquitin. (B) IF of endogenous p62 showing
preferential accumulation and persistence of p62 in
autophagy-defective cells. Bcl-2 expressing beclin1.sup.+/+,
beclin1.sup.+/-, atg5.sup.+/+ and atg5.sup.-/- iBMK cells were
stained for endogenous p62 under normal growth conditions (Day 0)
or following 7 days of metabolic stress (Day 7.sup.I) and 1 (Day
7+1.sup.R) and 2 (Day 7+1.sup.R) days of recovery. (C)
Autophagy-defective cells express constitutively higher levels of
exogenous myc-p62. Bcl-2 expressing atg5.sup.+/+ and atg5.sup.-/-
iBMK cells were transfected with myc-tagged p62 expression vector
(pcDNA3-myc-p62) and six independent cell lines of each were
evaluated for p62 expression levels by Western blotting with an
antibody recognizing the myc epitope tag.
[0023] FIG. 2 illustrates effects of metabolic stress in promoting
organelle damage and ER chaperones and PDI upregulation in
autophagy-deficient cells. (A-D) Representative electron
micrographs of Bcl-2 expressing atg5.sup.+/+ (A) or atg5.sup.-/-
(B) cells following 7 days of metabolic stress. A wild-type cell
(A) showing mitochondria (M, blue arrows and C, left panel), ER (E,
red arrows and C, right panel), mutant cell in (B) showing
mitochondria (M, blue arrows and D, left panel), and protein
aggregates (A, yellow and D, right panel). (E and F) 2-DIGE gels
showing differential regulation of ER chaperones (GRp170, GRp78),
PDI, PDI-P4H.beta. and ACO2 in Bcl-2 expressing atg5.sup.-/- iBMK
cells in response to metabolic stress (7 days). Total protein from
unstressed or metabolically stressed (7 days) Bcl-2 expressing
atg5.sup.+/+ and atg5.sup.-/- cell lines were labeled with Cy3
(Green-unstressed) or Cy5 (Red-stressed) and analyzed by 2-DIGE.
Images show 2-DIGE gels with proteins that are induced (Red),
repressed (Green) or unchanged (Yellow) under metabolic stress.
Protein spots (n=106) that were differentially induced were
selected and identified by mass spectrometry.
[0024] FIG. 3 illustrates the effects of autophagy defects in
altering the stress response and sensitize cells to ER stress and
proteasome inhibition. (A) Western blots showing levels of p62, ER
chaperones, PDI and ubiquitin in Bcl-2 expressing beclin1.sup.+/+,
beclin1.sup.+/-, atg5.sup.+/+ and atg5.sup.-/- iBMK cells following
5 or 7 days of metabolic stress, and 1 and 2 days of recovery.
(B-D) Allelic loss of beclin1 sensitizes cells selectively to ER
stress and proteasome inhibition. MTT assays showing sensitivity of
Bcl-2 expressing beclin1.sup.+/+ (Blue) and beclin1.sup.+/- (Red)
iBMK cells in response to increasing concentrations of (B)
tunicamycin for 3 days, or (C) epoxomycin for 2 days, and (D)
following 5 days of metabolic stress. Data are presented as
mean.+-.SD. (E) Autophagy deficiency causes mitochondrial damage.
Western blots showing levels of mitochondrial proteins (ACO2, LON,
SOD2 and PRDX3) in Bcl-2 expressing beclin1.sup.+/+,
beclin1.sup.+/-, atg5.sup.+/+ and atg5.sup.-/- iBMK cells,
following 0, 3 and 7 days of metabolic stress.
[0025] FIG. 4 illustrates elevated ER stress and DNA damage
response in autophagy-defective tumors. (A) Deficiency in atg5 in
iBMK cells promotes tumorigenesis. Tumor allograft growth of
atg5.sup.+/+ (red)), atg5.sup.+/- (yellow) and atg5.sup.-/- (blue)
iBMK cell lines in nude mice. (B) Deficiency in atg5 cooperates
with defective apoptosis enhances tumor growth. Tumor allograft
growth of Bcl-2 expressing atg5.sup.+/+ (red), and atg5.sup.-/-
(blue) iBMK cell lines in nude mice. (C) Western blot showing
elevated levels of p62 and ER chaperones and PDI in Bcl-2
expressing atg5.sup.+/+ and atg5.sup.-/- BMK tumors in (B). (D)
Elevated p62 and GRp170 levels in lung and heart tissues and
spontaneous lung tumors from beclin1.sup.+/- mice. Lung, heart and
spontaneous lung tumor sections from two 1.5-year-old
beclin1.sup.+/+ and four 1.5-year-old beclin1.sup.+/- mice were
stained for p62 and GRp170 by IHC. Stained sections were
independently scored and analyzed by Students t-test (lung) or
Mann-Whitney test (heart). p<0.05 was considered statistically
significant. (E) Elevated p62 and GRp170 in liver tissue and p62,
Mallory-Denk bodies (H&E, arrows) and .gamma.-H2AX (arrows) in
spontaneous liver tumors from beclin1.sup.+/- mice (1.5 years).
Stained sections were independently scored and analyzed by Students
t-test for significance (p<0.05). (F) Elevated p62, GRp170, and
.gamma.-H2AX positive nuclei in human hepatocellular carcinomas
(HCC). Representative images from a human liver TMA (46 samples)
showing H&E, p62, GRp170 and .gamma.-H2AX accumulation in HCCs.
Corresponding images from a normal human liver TMA (14 samples) are
shown for comparison. Stained sections were independently scored
and analyzed by Students t-test for significance for significance
(p<0.05).
[0026] FIG. 5 illustrates effects of metabolic stress in promoting
elevated ROS production and chromosomal instability in
autophagy-deficient cells. (A) Autophagy-deficiency leads to
elevated ROS production. Overlays show ROS levels in Bcl-2
expressing beclin1.sup.+/+ (Green) and beclin1.sup.+/- iBMK cells
(Blue) (x-axis, log scale) under normal growth (0Di) and at 0.5, 1,
2.5 and 24 hr (beclin1.sup.+/+, green arrows and beclin1.sup.+/-,
red arrows) during recovery (0.5-24 hR) from 5 days of metabolic
stress (5Di) by flow-cytometry using the ROS sensor DCF-DA. The ROS
levels in untreated cells are shown in dotted lines for comparison.
(B) Representative histogram from three independent experiments
measuring the mean ROS levels in Bcl-2 expressing beclin1.sup.+/+
and beclin1.sup.+/- iBMK cells shown in (A). (C) ROS scavenging
partially rescues the susceptibility to metabolic stress and
recovery due to allelic loss of beclin1. Representative time-lapse
images of Bcl-2 expressing beclin1.sup.+/+ and beclin1.sup.+/- iBMK
cells during recovery following 5 days of metabolic stress in
presence (NAC) and absence (UT) of the ROS scavenger NAC (1 mM)
(relative percentage of adherent cells compared to time 0 is
shown). (D) ROS scavenging suppresses p62 accumulation in
autophagy-deficient (beclin1.sup.+/- and atg5.sup.-/-) cells.
Western blot analysis of p62 levels in Bcl-2 expressing
beclin1.sup.+/+, beclin1.sup.+/-, atg5.sup.+/+ and atg5.sup.-/-
iBMK cell lines following 0 or 7 days of metabolic stress followed
by 1 day recovery without and with NAC. (E) ROS scavenging limits
progression to aneuploidy associated with allelic loss of beclin1.
Flow-cytometry analysis of DNA content in diploid, Bcl-2 expressing
beclin1.sup.+/+ and beclin1.sup.+/- iBMK cells grown in presence
(blue) or absence (red) of the ROS scavenger NAC (1 mM). Numbers
represent passage numbers at which ploidy was determined.
[0027] FIG. 6 illustrates the effects of failure to eliminate p62
by autophagy in activating the DNA damage response. (A) p62
expression leads to elevated ROS production in the
autophagy-defective beclin1.sup.+/- cells. Bcl-2 expressing
beclin1.sup.+/+ and beclin1.sup.+/- iBMK cells were transfected
with myc-tagged p62 or control vector and ROS levels (DCF-DA) were
measured by flow-cytometry at day 3 post-transfection. Histogram on
the right is representative of three independent experiments
measuring mean ROS level in each cell line on days 1 and 3
post-transfection. (B) Failure to eliminate p62 by autophagy under
metabolic stress leads to DNA damage response induction. Bcl-2
expressing atg5.sup.+/+ or atg5.sup.-/- iBMK cells stably
expressing EGFP or p62-EGFP were subjected to 3 days of metabolic
stress, allowed to recover for 1 day and stained for .gamma.-H2AX.
(C) Quantitation of the percentage cells with .gamma.-H2AX positive
foci in cells shown in (B), before and during recovery from 3 days
of metabolic stress. Data from 200 cells are presented as
mean.+-.SD. (D) Western blots showing RNAi-mediated knockdown of
p62 levels. Bcl-2 expressing wild-type (beclin1.sup.+/+ and
atg5.sup.+/+) and autophagy-defective (beclin1.sup.+/- and
atg5.sup.-/-) iBMK cells were transfected with either Lamin- or
p62-siRNA and subjected them to metabolic stress for 0, 24, 48 or
72 hr (24, 48, 72 and 96 hr post-transfection, respectively) and
total protein was analyzed for p62 levels. (E) p62 accumulation in
autophagy-defective cells is responsible for activation of the DNA
damage response. Bcl-2 expressing beclin1.sup.+/+, beclin1.sup.+/-,
atg5.sup.+/+ and atg5.sup.-/- iBMK cells (D), transfected with
either Lamin- or p62-siRNA were subjected to metabolic stress for
the indicated time and evaluated for .gamma.-H2AX positive nuclei.
(F) Quantitation of percentage cells with .gamma.-H2AX positive
nuclei from the data shown in (E). Data from two hundred cells are
presented as mean.+-.SD.
[0028] FIG. 7 illustrates how p62 expression cooperates with
autophagy-deficiency to promote tumor growth. (A) Western blot for
EGFP, in Bcl-2 expressing atg5.sup.+/+ and atg5.sup.-/- iBMK cells
stably expressing EGFP or p62-EGFP. (B) Tumor growth of cell lines
in (A) showing enhanced tumor growth in p62-EGFP expressing
atg5.sup.-/- tumors (red) compared to that of the control vector
(yellow). (C) Panel showing tumor-bearing mice injected with
p62-EGFP- (right panel), and EGFP-expressing (left panel)
atg5.sup.-/- iBMK cells from (B), at day 74 post-injection. (D)
EGFP fluorescence images of individual tumors from EGFP- and
p62-EGFP-expressing atg5.sup.-/- tumors. (E) Tumors from p62-EGFP
expressing atg5.sup.-/- cells are associated with p62 aggregates
and prevalent polymorphic and .gamma.-H2AX positive nuclei.
Representative photomicrographs of frozen tumor sections (left),
and paraffin embedded sections stained by H&E (middle) or IHC
for .gamma.-H2AX (right) in tumors from atg5.sup.-/- cells shown in
(C) and (D). (F) A model for the role of autophagy as a tumor
suppressor mechanism by limiting p62 accumulation.
[0029] FIG. 8 illustrates a cell-based screen for autophagy
inhibitors.
[0030] FIG. 9 illustrates a cell-based screen for rescue of
autophagy defect and p62 protein aggregate accumulation in response
to metabolic stress.
[0031] FIG. 10 illustrates reference images for a cell-based screen
for inhibitors of autophagy.
[0032] FIG. 11 illustrates reference images for a cell-based screen
for autophagy promoters.
[0033] FIG. 12 illustrates an overview of the autophagic pathway
and points of inhibition. Abbreviations: XL (Exelixis Vps34
inhibitors), HCQ (hydroxychloroquine)
DETAILED DESCRIPTION OF THE INVENTION
[0034] Embodiments of the present invention include methods of
screening for modulators of autophagy. In some embodiments,
cell-based screens may be used to identify activators and/or
inhibitors of autophagy. In certain embodiments, cell-based screens
may be used to identify genes whose expression modulates autophagy.
Such genes may represent targets for therapeutic intervention in
the autophagy pathway.
[0035] In one embodiment, the invention provides a method
comprising the steps of: (A) screening an shRNA library using a
test cell that expresses an autophagosome marker; (B) subjecting
the cell to metabolic stress; and C) performing analysis on the
cell to determine the localization of the autophagosome marker in
the test cell. Certain embodiments include step (D) comparing the
localization of the marker in the test cell with that of a control,
wherein a lower level of localization of the marker in
autophagosomes in the test cell compared to that demonstrated by
the control cell indicates knockdown of a gene whose expression
induces autophagy.
[0036] In another embodiment, the invention provides a method for
identifying inhibitors of autophagy comprising the steps of: (A)
contacting a test cell that expresses an autophagosome marker with
a compound; (B) subjecting the test cell to metabolic stress; and
(C) performing analysis on the test cell to determine the
localization of the autophagosome marker in the cell. Certain
embodiments include step (D) comparing the localization of the
marker in the cell with that of a control cell, wherein a lower
level of localization of the marker in autophagosomes in the test
cell compared to that demonstrated by the control cell indicates
that the compound is capable of inhibiting autophagy.
[0037] In certain embodiments, cells used in the methods may be
apoptosis-defective. In certain preferred embodiments, the cells
may be bax.sup.-/-bak.sup.-/-.
[0038] In certain embodiments, the autophagosome marker comprises
EGFP-LC3. In preferred embodiments, the analysis performed
comprises image analysis to determine a level of punctate
localization of the autophagosome marker.
[0039] In another embodiment of the invention, a method is provided
comprising the steps of: (A) screening an shRNA library using an
autophagy-defective test cell expressing a marker of protein
aggregation; (B) subjecting the test cell to metabolic stress; and
(C) performing analysis on the test cell to determine the level of
the marker. Certain embodiments include step (D) comparing the
level of the marker in the test cell with that of a control cell,
wherein a lower level of aggregates comprising the marker in the
test cell compared to that demonstrated by the control cell
indicates the lowered expression of a gene whose knockdown
increases aggregate clearance.
[0040] Another embodiment is directed to a method for identifying
stimulators of autophagy comprising the steps of: (A) contacting a
test cell expressing a marker of protein aggregation with a
compound; (B) subjecting the cell to metabolic stress; and (C)
performing analysis on the test cell to determine the level of the
marker. Certain embodiments include step (D) comparing the level of
the marker in the test cell with that of a control cell, wherein a
lower level of aggregates comprising the marker in the test cell
compared to that demonstrated by the control cell indicates that
the compound is capable of increasing autophagy.
[0041] In certain embodiments, cells used in the methods may be
apoptosis-defective. In certain embodiments, the cells may be
autophagy-defective. Preferably, cells may have reduced expression
of one or more of the Beclin1, Atg5, or Atg7 genes.
[0042] In certain embodiments, the marker of protein aggregation
comprises p62 protein linked to a label molecule. In preferred
embodiments, the label molecule is enhanced green fluorescent
protein (EGFP). In certain embodiments, the analysis performed
comprises image analysis to determine the level of p62 protein
aggregates.
[0043] In preferred embodiments of the invention, cells useful in
methods for screening for modulators of autophagy include, without
limitation, immortalized baby mouse kidney cells.
[0044] In certain embodiments, cell-based methods according to the
invention may be used for high-throughput screening assays.
[0045] It has now been found that metabolic stress caused
autophagy-defective tumor cells to preferentially accumulate p62,
ER chaperones such as glucose-regulated protein 170 (GRp170),
glucose-regulated protein 78 (GRp78), calnexin and protein
disulphide isomerases (PDIs), indicative of protein quality control
failure. Moreover, autophagy defects caused mitochondrial
destruction, elevated oxidative stress and activation of the DNA
damage response, which were attributed directly to persistence of
p62. The failure to degrade p62 and the resulting cytotoxic effects
manifested during stress and recovery with defective autophagy,
were partially suppressed by ROS scavengers indicating that
persistence of p62 and oxidative stress contributed to cellular
damage. This failure to clear p62 following stress in
autophagy-defective cells greatly accelerated tumorigenesis,
indicating that autophagy is required to prevent p62 accumulation,
that if allowed to persist, is sufficient to promote tumor growth.
p62 is commonly upregulated in human tumors, suggesting that
defective autophagy is a mechanism by which this occurs and that is
an oncogenic mechanism.
[0046] Inactivation of the autophagy tumor suppressor mechanism is
common in tumors, not only through allelic loss of beclin1, but
also potentially through indirect mechanisms such as constitutive
activation of the PI-3 kinase pathway, which activates mTOR that
suppresses autophagy. In tumor cells with Rb and p53 checkpoints
inactivated, defects in autophagy impair stress tolerance and
amplify genome damage, suggesting that the protective function of
autophagy limits genetic instability and suppresses tumor
initiation and progression. While not intending to be limited by
any theory or theories of operation, it is believed that the
increased damage that results from the inability of
autophagy-defective cells to manage metabolic stress may facilitate
tumor initiation and progression of those cells that do not die. If
so, then the tumor suppression mechanism of autophagy is related to
how this management of metabolic stress prevents cellular
damage.
[0047] Evidence from model organism disease models indicates that
promoting autophagy with mTOR inhibitors such as rapamycin, and
enhancing the clearance of misfolded, damaged or mutated proteins
and protein aggregates prevents neurodegeneration, but that there
also are mTOR independent means to increase autophagy. Similarly,
genetically eliminating the expression of p62 in hepatocytes and
preventing p62 accumulation in autophagy-defective atg7.sup.-/-
hepatocytes dramatically suppresses the phenotype of
steatohepatitis. In contrast, neurodegeneration due to expression
and accumulation of polyglutamate expansion mutant proteins is
greatly exacerbated by allelic loss of beclin1 and defective
autophagy. Thus, autophagy may be required to limit the buildup of
misfolded, mutated proteins in p62-containing protein aggregates,
which leads to cellular deterioration and disease.
[0048] Autophagy-defective mouse tissues have low ATP levels and
accumulate polyubiquitinated- and p62-containing protein aggregates
and abnormal mitochondria, indicating failure of energy homeostasis
and protein and organelle degradation. This may be associated with
elevated cell death and persistence of dead cells that cannot be
engulfed or degraded. It has now been found that accumulation of
p62 in response to metabolic stress represents a striking phenotype
of autophagy-defective tumors cells with allelic loss of beclin1 or
deficiency in atg5, suggesting defective protein quality control is
potentially a major contributing factor to tumorigenesis. Thus
autophagy appears to be a mechanism by which tumor cells rapidly
and efficiently turnover p62.
[0049] Unlike that seen in mouse brain tissue, there is no striking
accumulation of polyubiquitinated proteins in tumor cells with
defective autophagy, suggesting that there may be tissue-specific
differences in autophagy-mediated protein elimination (FIG. 4C).
Dilution of polyubiquitinated proteins through active cell division
in tumor cells may prevent their accumulation, which is not
possible in post-mitotic neurons. Alternatively,
proteasome-mediated turnover of polyubiquitinated proteins may be
elevated in tumor cells in comparison to neuronal tissues. Indeed,
autophagy defects sensitize cancer cells to proteasome inhibitors,
suggesting a compensatory function of the two protein degradation
pathways.
[0050] The persistence of p62 and p62-containing protein aggregates
in beclin1 and atg5 mutant cells and tumors indicated a profound
defect in the management of protein turnover supported by the
associated accumulation of ER chaperones and the oxidative protein
folding machinery. This suggested that the inability to degrade
damaged or misfolded proteins through autophagy in stressed cells
increased the burden on the ER chaperones and oxidative protein
folding machinery, necessitating their upregulation. Both p62 and
GRp170 were dramatically upregulated in beclin1.sup.+/- tissues as
well as in spontaneous tumors, indicating that coping with unfolded
proteins may be a biomarker for impaired autophagy that precedes
tumor initiation.
[0051] ER chaperone and PDI upregulation is common in human tumors,
and increased GRp170 expression is associated with poor prognosis
in breast cancer. Chaperones are stress-responsive and provide a
protective function by suppressing the accumulation of unfolded
proteins that may be a particularly important compensatory
mechanism for autophagy-defective cells. It is important to note
that an increased demand for protein folding can be a source of
oxidative stress, particularly when cells are overburdened with
damaged and unfolded proteins, in concordance with increased ROS in
stressed beclin1.sup.+/- cells. Interestingly, metabolic stress
induces HIF-1.alpha. in vitro and in tumors in vivo, and many of
the proteins that are induced by metabolic stress and
preferentially elevated in autophagy-deficient cells are targets of
HIF-1.alpha.. While HIF-1.alpha. induction promotes autophagy, this
may play a role as a negative feedback loop to curtail HIF-1.alpha.
activation.
[0052] Autophagy-defective tumor cells subjected to stress display
accumulation of damaged mitochondria as an additional source of
oxidative stress. Thus, the accumulation of unfolded protein and
protein aggregates and the persistence of damaged mitochondria may
collectively lead to elevated ROS production in stressed,
autophagy-defective cells. As ROS scavengers partially suppress p62
accumulation and cell death in stressed beclin1.sup.+/- tumor
cells, this suggests that the elevated oxidative stress contributes
to cell damage and death. Stress-mediated p62 protein accumulation
in autophagy-defective cells is sufficient for ROS and DNA damage
response induction, whereas wild-type cells eliminated p62 and does
not induce ROS or the DNA damage in response to stress. The ability
to rescue .gamma.-H2AX-positive nuclear staining in stressed,
autophagy-defective cells with knockdown of p62 indicates that this
elevated activation of the DNA damage response was attributable
directly to p62 accumulation. Thus, the inability of
autophagy-defective tumor cells to eliminate p62 contributes to
oxidative stress and likely to DNA damage. These observations are
strikingly similar to the rescue of oxidative stress toxicity
caused by atg7 deficiency with p62 deficiency in mouse liver
(Komatsu, M., et al. (2007), Homeostatic Levels of p62 Control
Cytoplasmic Inclusion Body Formation in Autophagy-Deficient Mice,
Cell 131, 1149-1163). In normal tissues, toxicity due to p62
accumulation resulting from autophagy defect may appropriately
trigger cell death, whereas in checkpoint-defective tumor cells
this instead may result in enhancement of genome instability,
mutations and tumor progression.
[0053] atg5.sup.-/- tumors display pronounced p62 and p62 aggregate
accumulation and this p62 expression is sufficient for a remarkable
enhancement of tumor growth. Moreover, p62-EGFP but not
EGFP-expressing atg5.sup.-/- tumors display prevalent polyploid and
aneuploid nuclei and are positive for .gamma.-H2AX, indicative of
DNA double strand breaks. This indicates that p62 persistence in
autophagy-defective cells is sufficient for induction of DNA damage
and genome instability associated with enhanced tumor growth. p62
expression, p62 aggregates, and Mallory-Denk bodies that are
composed of p62 and other proteins are common in steasosis and in
hepatocellular carcinomas and other cancers. Defects in autophagy
may be a major mechanism for sustained p62 accumulation and
formation of Mallory-Denk bodies. Importantly, it has been shown
now that p62 accumulation is not merely a histologic marker for
certain cancers, but rather, directly contributes to tumor growth.
Examination of a human liver cancer TMA reveals the high frequency
of p62, GRp170 and .gamma.-H2AX positive cells similar to what was
observed in the autophagy-defective tumor allografts and
spontaneous liver tumors arising in beclin1.sup.+/- mice. Thus
autophagy defects, p62 and ER chaperone accumulation, and DNA
damage are common, associated tumor phenotypes. Interestingly,
pten, beclin1, or atg7 deficiency produce HCC and liver steatosis
in mice, suggesting that constitutive activation of the PI3-kinase
pathway and the resulting steatosis may be caused by suppression of
autophagy.
[0054] p62 normally functions as an adaptor protein by binding to
TRAF6 in cell surface receptor signaling complexes that regulate
the activation of NF-.kappa.B. This function of p62 is also
required for efficient oncogene activation in vitro and p62
deficiency suppresses spontaneous lung tumorigenesis by K-ras.
Thus, p62 had been identified as an oncoprotein in both loss- and
gain-of-function situations (FIG. 7). Alternatively, liver-specific
autophagy defects produce p62 accumulation that causes oxidative
stress and activation of Nrf2. Thus, the tumor suppressor function
of autophagy may be related to the modulation of oxidative stress,
Nrf2 or possibly also HIF-1.alpha., and NF-.kappa.B pathways. While
not intending to be bound by any theory or theories of operation,
it is believed that defects in autophagy promote a failure of
protein and organelle quality control in tumors that leads to
oxidative stress, genome damage and tumorigenesis caused by the
inability to eliminate p62 following stress. As p62 upregulation is
common in liver tissues of individuals at risk and hepatocellular
carcinomas in patients, this suggests that facilitating the
clearance of p62 by promoting autophagy may be a strategy for
cancer chemoprevention.
[0055] Allelic loss of the essential autophagy gene beclin1 occurs
in human cancers and renders mice tumor-prone, suggesting that
autophagy is a tumor-suppression mechanism. While tumor cells
utilize autophagy to survive metabolic stress, autophagy mitigates
the resulting genome damage, thus functioning to limit
tumorigenesis. In response to stress, autophagy-defective tumor
cells preferentially accumulate p62/SQSTM1 (p62) protein
aggregates, endoplasmic reticulum (ER) chaperones, damaged
mitochondria, reactive oxygen species (ROS), and genome damage.
Thus, autophagy may suppress oxidative stress and protein,
organelle and DNA damage. Suppressing ROS or p62 accumulation
provided protection from damage resulting from autophagy defects.
Moreover, stress-mediated p62 accumulation caused by defective
autophagy stimulates ROS and the DNA damage response and promotes
tumorigenesis. These findings suggest that the tumor-suppressive
function of autophagy is related to the prevention of sustained p62
accumulation, oxidative damage and genome instability that enable
tumor progression. While not intending to be limited by any theory
or theories of operation, defective autophagy is believed to be a
mechanism for p62 upregulation commonly observed in human tumors
that contributes directly to tumorigenesis.
[0056] Stimulating autophagy may be critically important to limit
the progression of certain diseases, including, but not limited to,
neurodegeneration, liver disease, and also cancer, by facilitating
the elimination of protein aggregates, damaged organelles, and the
toxic consequences of their accumulation. In individuals at risk
for hepatocellular carcinoma (HCC), autophagy stimulators may be
particularly indicated to limit liver damage and disease
progression for cancer chemoprevention. Thus, the autophagy pathway
provides a basis for novel therapeutic target identification for
drug discovery for many diseases with respect to both acute
treatment and also disease prevention.
[0057] Identification of the therapeutic means to inhibit the
autophagy survival pathway in tumor cells would be advantageous.
This may be especially important as many therapeutics currently in
use, such as kinase and angiogenesis inhibitors, inflict metabolic
stress, which increases the dependency on autophagy for survival.
Furthermore, tumor cells with impaired autophagy are particularly
vulnerable to metabolic stress and further therapeutic suppression
of autophagy may exploit this vulnerability by promoting cell death
by metabolic catastrophe or the failure to mitigate cell damage
accumulation. In support of these hypotheses, preclinical findings
using hydroxychloroquine (HCQ) to inhibit lysosome acidification
and thereby autophagy in combination therapy have been described
(FIG. 12). Specific inhibitors of the autophagy survival pathway in
tumor cells, including, without limitation, Vps34 inhibitors
(available from Exelixis), may be used in combination with agents
such as for example, angiogenesis and kinase inhibitors that
promote metabolic stress (FIG. 12). In contrast to most currently
available therapies that target proliferating tumor cells,
autophagy inhibition may be used to target nondividing and
potentially even dormant tumor cells.
[0058] To address the need for the identification of autophagy
inhibitors for cancer prevention and/or treatment and autophagy
stimulators for the prevention and treatment of disease, cell-based
screens are described. Specific embodiments include shRNA screens
as well as small molecule screens.
[0059] In certain embodiments, a small hairpin RNA (shRNA) screen
may be used. shRNA screens may utilize, without limitation, kinase,
phosphatase, and/or metabolism shRNA lentivirus library subsets. In
certain embodiments, lentivirus libaries may include approximately
5000 genes in triplicate. Lentiviruses are high-titer, individual
clones with representation of five independent hairpins for each
target gene supplied in a high-throughput format (Root, D. E.,
Hacohen, N., Hahn, W. C., Lander, E. S., and Sabatini, D. M.
(2006), Genome-scale loss-of-function screening with a lentiviral
RNAi library, Nature Methods 3, 715-719.).
[0060] In other embodiments of the invention, small molecule
screens may be used. Such assays may utilize chemical libraries.
Useful libraries include, but are not limited to, the National
Cancer Institute (NCl) training set, Diversity set, Maybridge, and
the Main library. Fluorescence image analysis may be used to
capture the data. Positive clones may be subject to subsequent
independent verification.
[0061] According to certain embodiments, useful cell lines for the
screens include genetically defined immortal mouse epithelial cell
lines derived from wild type or autophagy deficient mice with
well-characterized phenotypes in vitro and in vivo (Degenhardt et
al., 2006; Mathew et al., 2007b; Karantza-Wadsworth et al., 2007).
In preferred embodiments, useful cell lines that may be employed in
the described screens include cells having the phenotype(s)
including, but not limited to, beclin1.sup.+/-, atg5.sup.-/-,
and/or atg7.sup.-/-. Cell may be derived from multiple tissues.
Preferably, the cell lines are derived from mouse tissues. In
certain preferred embodiments, the cell line comprises immortalized
baby mouse kidney epithelial cells.
[0062] Genes and compounds identified in the screens may be
validated in vitro and/or in vivo. Tissues and spontaneous tumors
from mouse models may be collected for validation purposes.
Conditional knockouts may also be used for validation.
EXAMPLES
[0063] The following examples serve to more fully describe the
manner of using the above-described invention. It is understood
that these examples in no way serve to limit the true scope of this
invention, but rather are presented for illustrative purposes.
Materials and Methods
Generation of Stable Cell Lines and Culture Conditions
[0064] Primary epithelial cells from atg5.sup.+/+, atg5.sup.+/-,
atg5.sup.-/-, beclin1.sup.+/+ and beclin1.sup.+/- mice were
immortalized to generate iBMK cell lines (Degenhardt, K., et al.
(2006), Autophagy promotes tumor cell survival and restricts
necrosis, inflammation, and tumorigenesis, Cancer Cell 10, 51-64;
Degenhardt, K., et al., (2002) Bax and Bak independently promote
cytochrome C release from mitochondria, J Biol Chem 277,
14127-14134; Mathew, R., Degenhardt, K., Haramaty, L., Karp, C. M.,
and White, E. (2008), Immortalized mouse epithelial cell models to
study the role of apoptosis in cancer, Methods Enzymol, 446,
77-106; Mathew, R., Kongara, S., Beaudoin, B., Karp, C. M., Bray,
K., Degenhardt, K., Chen, G., Jin, S., and White, E. (2007b),
Autophagy suppresses tumor progression by limiting chromosomal
instability, Genes Dev 21, 1367-1381). Bcl-2 expressing
atg5.sup.+/+ and atg5.sup.-/- iBMK cells were engineered to stably
express myc-tagged p62 (pcDNA3-myc-p62), EGFP (pEGFPC1) or p62-EGFP
(pEGFPC1-p62) (Rodriguez, A., et al. (2006), Mature-onset obesity
and insulin resistance in mice deficient in the signaling adapter
p62, Cell Metab 3, 211-222), co-transfected with pcDNA3-Zeo by
electroporation (Nelson, D. A., et al. (2004) Hypoxia and defective
apoptosis drive genomic instability and tumorigenesis, Genes Dev
18, 2095-2107). Independent clones were selected in zeocin (500
.mu.g/mL) and expanded as stable cell lines in normal culture
conditions (DMEM, 10% FBS, 1% Pen Strep (Invitrogen, Carlsbad,
Calif.) at 38.5.degree. C. and 8.5% CO.sub.2) (Mathew, R.,
Degenhardt, K., Haramaty, L., Karp, C. M., and White, E. (2008)).
To induce metabolic stress, cells were placed in glucose-free DMEM
(Invitrogen) containing 10% FBS and incubated with a defined gas
mixture containing 1% oxygen, 5% CO.sub.2 and 94% N.sub.2
(GTS-Welco, Allentown, Pa.) (Nelson et al., 2004). NAC
(Sigma-Aldrich, St. Louis, Mo.) was used at a concentration of 1
mM.
Example 1
Autophagy-Defective Tumor Cells Accumulate p62 in Response to
Stress
[0065] To address the potential role of autophagy-dependant protein
quality control in tumor suppression, p62 modulation was assessed
during metabolic stress and recovery in autophagy-competent
(beclin1.sup.+/+ and atg5.sup.+/+) and autophagy-defective
(beclin1.sup.+/- and atg5.sup.-/-) immortalized baby mouse kidney
(iBMK) cells. Cells were engineered to express Bcl-2, as the
assessment of autophagy is facilitated in an apoptosis-defective
background (Degenhardt, K., et al. (2006), Autophagy promotes tumor
cell survival and restricts necrosis, inflammation, and
tumorigenesis, Cancer Cell 10, 51-64; Lum, J. J., et al., (2005),
Growth factor regulation of autophagy and cell survival in the
absence of apoptosis, Cell 120, 237-248). Under normal growth
conditions, p62 levels were low in wild-type cells and slightly
elevated in autophagy-defective iBMK cells (FIG. 1B). Following 7
days of metabolic stress there was dramatic p62 induction in
wild-type cells that was further elevated in autophagy-defective
cells in a predominantly punctate pattern suggestive of p62
aggregation. In wild-type cells, most p62 aggregates were
eliminated within 24 hr of recovery, whereas p62 remained
predominantly in large aggregates in autophagy-defective cells
(FIG. 1B). p62 aggregates persisted in mutant cells after 2 days of
recovery (FIG. 1B) and remained so for at least a week (data not
shown), indicating that autophagy is required to limit the
formation and to promote the clearance of p62. Consistent with
this, higher p62 levels were observed in autophagy-deficient
(atg5.sup.-/-), as compared to wild-type (atg5.sup.+/+) iBMK cells
stably expressing myc-tagged p62 (myc-p62) (FIG. 1C). Thus,
metabolic stress induced p62 accumulation and aggregate formation,
requiring autophagy for elimination.
Example 2
Autophagy-Defective Tumor Cells Accumulate Damaged Mitochondria ER
Chaperones and PDIs
[0066] Apoptosis-deficient atg5.sup.+/+ iBMK cells responded to
prolonged stress by undergoing progressive autophagy, yielding
cells less than one-third their original size that retained
numerous well-preserved mitochondria (M), and ER appeared slightly
distended (E) indicative of an unfolded protein response (UPR)
(FIGS. 2A and 2C). In contrast, Bcl-2-expressing atg5.sup.-/- (FIG.
2B) and beclin1.sup.+/- (data not shown) iBMK cells showed
disintegrating mitochondria with gross structural abnormalities (M)
and large, abnormal cytoplasmic structures (A) resembling protein
aggregates (FIGS. 2B and 2D), consistent with p62 aggregate
accumulation (FIG. 1B). Thus, autophagy may function to prevent the
accumulation of protein aggregates and damaged organelles during
metabolic stress.
[0067] Since tumor cells with defective autophagy displayed failure
of protein quality control, two-dimensional difference in gel
electrophoresis (2-DIGE) coupled with mass spectrometry were
performed to characterize the impact on the cellular proteome.
Autophagy-competent, apoptosis-defective (bax.sup.-/-/bak.sup.-/-)
D3 iBMK cells manage long-term metabolic stress by activation of
autophagy. In response to metabolic stress, D3 cells induced ER
chaperones (GRp170, GRp78, calreticulin), PDIs metabolism and
mitochondrial proteins (FIGS. 2C and 2D). Some of these proteins
(triosphosphate isomerase-1 [TPI-1]; phosphoglycerate kinase
1[PGK-1]; pyruvate kinase 3 [PK-3]; glycerol-3-phosphate
dehydrogenase [GPDH]) are targets of hypoxia inducible factor-1
.alpha. (HIF-1.alpha.) indicative of metabolic adaptation, and
HIF-1.alpha. is induced in iBMK cells by metabolic stress in vitro
and in tumors in vivo. Similarly to D3 cells under metabolic stress
proteomic analysis of Bcl-2 expressing autophagy-competent
beclin1.sup.+/+ and atg5.sup.+/+ iBMK cells induced ER chaperones
GRp170, GRp78, calreticulin and calnexin indicating that metabolic
stress response was not influenced by the means of apoptosis
inactivation. To determine if autophagy-deficiency altered this
stress response, Bcl-2 expressing, apoptosis and
autophagy-defective (beclin1.sup.+/- and atg5.sup.-/-) iBMK cells
were examined by 2-DIGE in parallel. Autophagy-defective cells
displayed preferential upregulation of ER chaperones compared to
the wild-type cells (FIGS. 2E and 2F). Allelic loss of beclin1 was
associated with marked differential increase in GRp170, GRp78,
calreticulin and calnexin while atg5.sup.-/- cells showed
differential increase in GRp170, GRp78 and calnexin compared to the
wild-type cells. For example, GRp170 levels were induced by 3- to
almost 9-fold in autophagy-competent (D3, beclin1.sup.+/+ and
atg5.sup.+/+) cells whereas induction was more than 8-fold in
autophagy-deficient (beclin1.sup.+/- and atg5.sup.-/-) cells under
metabolic stress (FIG. 2F). A similar but less striking
differential increase was also observed in GRp78 levels in
beclin1.sup.+/- and atg5-/- cells. Members of the PDI family of
proteins such as PDI and PDI-prolyl 4-hydroxylase-.beta. subunit
(PDI-P4H.beta.) instrumental in refolding misfolded proteins in the
ER lumen, were induced under metabolic stress and were further
elevated by autophagy-deficiency (FIGS. 2E and 2F). It is
hypothesized that the lack of induction of calreticulin and
PDI-P4H.beta. by metabolic stress in atg5.sup.-/- iBMK cells, may
be due to their increased susceptibility to metabolic stress.
Interestingly, levels of cytoskeletal and protein synthesis-related
proteins were repressed with stress in all cell lines. This
differential induction of ER chaperones and oxidative protein
folding machinery in the autophagy-deficient cells under stress
suggests a role for autophagy in mitigating ER stress by
eliminating unfolded proteins through lysosomal degradation.
[0068] Individual proteins and their degradation fragments can be
represented by multiple spots in 2-DIGE and this complicates
estimation of protein levels by spot volume ratios. Thus, ER
chaperones identified as differentially regulated by 2-DIGE were
further examined by Western blotting. p62 and ER chaperones GRp
170, GRp78, calnexin and PDI, and showed higher induction in
beclin1.sup.+/- and atg5.sup.-/- compared to beclin1.sup.+/+ and
atg5.sup.+/+ cells under stress (FIG. 3A) consistent with p62
immunofluorescence (IF) (FIG. 1B) and proteomic analysis. As with
p62 aggregates, elevated and persistent levels of these proteins
were more evident in atg5.sup.-/- and beclin1.sup.+/- cells during
1-2 days of recovery from metabolic stress (FIG. 3A). Together,
these results suggested that autophagy defects accentuated the
demand for protein folding under metabolic stress that persists
during recovery.
Example 3
Autophagy Defects Cause Sensitivity to ER Stress
[0069] Since autophagy defects indicated an elevated demand for
protein folding, the sensitivity of beclin1 cells to
pharmacological induction of ER stress was investigated.
Tunicamycin induces ER stress by inhibiting protein glycosylation
and allelic loss of beclin1 caused increased sensitivity to this
drug when compared to wild-type cells (FIG. 3B). Degradation of
unfolded proteins via the proteasome pathway and ameliorates ER
stress and to investigate if autophagy deficiency also elevated the
burden on the ubiquitin-proteasome system, the sensitivity to the
proteasome inhibitor epoxomycin was assessed. Autophagy-defective
cells displayed increased sensitivity to proteasome inhibition,
which was further exacerbated by metabolic stress (FIGS. 3C and
3D). This suggests an increased dependency of autophagy-defective
cells on proteasome pathway particularly during metabolic stress.
Thus, autophagy may function to maintain protein quality control
cooperatively with the ubiquitin-proteasome pathway, consistent
with the observation that suppression of proteasome function
activates autophagy, the inhibition of which promotes cell death
(Ding, W. X., et al., (2007), Linking of autophagy to
ubiquitin-proteasome system is important for the regulation of
endoplasmic reticulum stress and cell viability, Am J Pathol 171,
513-524).
[0070] EM revealed preferential accumulation of morphologically
abnormal mitochondria in autophagy-deficient cells (FIGS. 2A-2D)
and stressed atg5.sup.-/- cells showed aberrant regulation of
mitochondrial proteins such as aconitase (ACO2) (FIGS. 2E and 2F)
consistent with mitochondrial deterioration. ACO2 is a
mitochondrial ROS sensor that responds to, and accumulates under,
mitochondrial oxidative stress whereupon it either aggregates or is
degraded by its specific protease LON. Consistent with the profound
induction of stress proteins and mitochondrial damage,
autophagy-deficient cells showed markedly increased
(beclin1.sup.+/-) or reduced (atg5.sup.-/-) levels of ACO2 after 7
days of metabolic stress compared to the wild-type cells (FIG. 3E).
Furthermore, LON, which is responsible for the degradation of the
oxidized form of ACO2, showed progressively increasing levels under
metabolic stress, as was also observed for oxidative stress markers
such as superoxide dismutase (SOD2) and peroxiredoxin (PRDX3) that
were not sustained in autophagy-defective cells (FIG. 3E). These
results suggested that autophagy defects are associated with
mitochondrial deterioration under metabolic stress.
Example 4
Defects in Autophagy Cause Upregulation of p62 and ER Chaperones in
Tumors
[0071] To assess whether the differential accumulation of p62, ER
chaperones and PDI was also a feature of autophagy defects in
tumors, Bcl-2 expressing atg5.sup.+/+ and atg5-/-, and
beclin1.sup.+/+ and beclin1.sup.+/- iBMK tumor allografts, and
spontaneous tumors from beclin1.sup.+/- mice were examined. As with
autophagy defects caused by allelic loss of beclin1 in iBMK cells,
deficiency in atg5 increased tumorigenesis and cooperated with
defects in apoptosis to accelerate tumor growth (FIG. 4B). All
Bcl-2 expressing atg5.sup.-/- tumors displayed elevated p62,
GRp170, GRp78, calnexin, and PDI compared to the wild-type by
Western blotting (FIG. 4C) as did Bcl-2 expressing beclin1.sup.+/-
and atg5.sup.-/- tumors by immunohistochemistry (IHC) (data not
shown). Upregulation of ubiquitinated proteins, although common in
tissues of autophagy-defective mice was less striking in
atg5.sup.-/- tumors (FIG. 4C) and may reflect the rapidly dividing
state of these cells. Additionally, histological analyses of
tissues (lung, heart, and liver) and spontaneous lung and liver
tumors from 1.5 year-old beclin1.sup.+/- mice showed significant
accumulation of p62 (lung, p<0.029, t-test; heart, p<0.025,
Mann-Whitney test) and GRp170 (lung, p<0.0001, t-test; heart,
p<0.028, Mann-Whitney test) when compared with age-matched
tissues from beclin1.sup.+/+ littermates (FIGS. 4D and 4E). This
suggested that failure of autophagy-mediated protein degradation
due to allelic loss of beclin1 in vivo caused elevated ER chaperone
levels as a compensation mechanism when proteins destined for
degradation were not eliminated through autophagy and that this
phenotype is manifested in tissues and spontaneous tumors.
[0072] p62 and ER chaperone upregulation are common in human tumors
and are often associated with poor prognosis. Human hepatocellular
carcinoma (HCC) in particular, is associated with dramatic p62
accumulation in Mallory-Denk bodies, and beclin1.sup.+/- mice
display p62 accumulation in liver in association with
steatohepatitis and spontaneous liver tumors (FIG. 4E), suggesting
that autophagy defects may play a prominent role in HCC etiology.
Indeed, liver and spontaneous liver tumors from beclin1.sup.+/-
mice also showed significantly higher levels of p62 (p<0.002,
t-test), GRp170 (p<0.003, t-test) and DNA damage response
activation (.gamma.-H2AX positive nuclei) (p<0.014, t-test)
compared to the normal liver tissues from age-matched
beclin1.sup.+/+ mice (FIG. 4E). Therefore, p62, GRp 170 and the DNA
damage response (.gamma.-H2AX) were examined in a panel of human
liver and HCCs from a liver tissue microarray (TMA) by IHC. Indeed,
p62 (p<0.0001, t-test) and GRp170 (p<0.0001, t-test) were
significantly upregulated with high frequency in HCC compared with
normal liver (FIG. 4F). Moreover, as with spontaneous liver tumors
in beclin1.sup.+/- mice (FIG. 4E), human HCCs were also associated
with higher levels of .gamma.-H2AX positive nuclei (p<0.03,
t-test) compared to normal liver samples (FIG. 4F). These results
suggest that, as with stressed autophagy-deficient cells,
accumulation of p62 and GRp170 in human tumors may be a common
symptom of defective autophagy manifesting as accumulation of
unfolded proteins associated with activation of the DNA damage
response. Moreover, failure of protein and organelle quality
control caused by autophagy defects may cause elevated oxidative
stress and DNA damage that may be genotoxic.
Example 5
Autophagy Mitigates Oxidative Stress and Progression to
Aneuploidy
[0073] Activation of the DNA damage response is a hallmark of
oxidative stress that can be elevated due to increased ROS levels.
Protein re-folding in the ER by PDIs can enhance oxidative stress
through redox reactions involving free radicals. Additionally, the
mitochondrial stress and damage frequently observed in
autophagy-deficient cells (FIGS. 2 and 3 can be a potential source
of ROS. Since autophagy deficiency preferentially causes the
accumulation of p62 aggregates, damaged mitochondria and
upregulation of oxidative protein folding machinery under metabolic
stress and is associated with activation of the DNA damage response
in tumors, the ROS levels in beclin1.sup.+/+ and beclin1.sup.-/-
cells was examined.
[0074] Under normal growth conditions ROS levels were slightly
elevated in the beclin1.sup.+/- iBMK cells compared to the
wild-type cells, however, following 5 days of metabolic stress
there was a marked increase in beclin1.sup.+/- cells (FIGS. 5A and
5B). During recovery, ROS levels either remained unchanged or
slightly increased in the beclin1.sup.+/+ iBMK cells and returned
to normal levels by 24 hr whereas, beclin1.sup.+/- iBMK showed a
marked increase in ROS levels most apparent after 1 hr that
persisted following 24 hr of recovery (FIGS. 5A and 5B). These
results indicated that allelic loss of beclin1 was associated with
elevated and persistent ROS production throughout metabolic stress
and recovery.
[0075] To determine if the elevated ROS and oxidative stress in
autophagy-deficient cells contributes to cellular damage, the
stress response without and with the ROS scavenger, N-acetyl
cysteine (NAC) was examined. Following 5 days of metabolic stress,
Bcl-2 expressing beclin1.sup.+/- iBMK cells showed increased
susceptibility to stress compared to wild-type cells (FIG. 5C). The
presence of NAC during metabolic stress improved survival, and this
protective effect was more profound in beclin1.sup.+/- cells,
suggesting that elevated induction and poor management of ROS
levels are partly responsible for the increased susceptibility of
autophagy-defective cells to metabolic stress (FIG. 5C). This
enhanced survival provided by NAC was associated with decreased p62
accumulation during metabolic stress in the beclin1.sup.+/- iBMK
cells (FIG. 5D), suggesting that ROS-mediated oxidative stress can
lead to protein damage and accumulation of p62 aggregates. A key
feature of genomic instability associated with autophagy defects is
the accelerated progression to aneuploidy under normal culture
conditions. To examine the role of increased ROS levels and
oxidative stress on genomic instability, the DNA content of early
passage diploid beclin1.sup.+/+, and autophagy-deficient
beclin1.sup.+/- iBMK cells without and with NAC by flow-cytometry
were monitored. beclin1.sup.+/+ iBMK cells maintained diploidy
after 40 passages and the presence of NAC had no effect. In
contrast, beclin1.sup.+/- cells showed accelerated progression to
aneuploidy by passages 18 and 39 (FIG. 5E), and NAC delayed this
progression (FIG. 5E), indicating a causative role for persistent
basal ROS-mediated oxidative stress in progression to aneuploidy
associated with autophagy defects. Thus, metabolic stress causes
p62 accumulation mediated in part by elevated ROS production and
the failure to suppress this ROS accumulation in
autophagy-deficient cells is associated with genomic
instability.
Example 6
p62 Accumulation is Sufficient to Activate the DNA Damage
Response
[0076] Since autophagy deficiency leads to accumulation of p62,
oxidative stress and accelerated progression to aneuploidy, it was
examined whether accumulation of p62 was sufficient to induce ROS
and the DNA damage response. Transient expression of p62-EGFP
formed aggregates and was sufficient to elevate ROS levels in the
autophagy-deficient (beclin1.sup.+/- and atg5.sup.-/-), but not in
the wild-type cells, whereas EGFP expression alone did not induce
ROS in either (FIG. 6A). Transient p62-EGFP expression was also
associated with DNA damage response activation (.gamma.-H2AX
positive nuclei) in autophagy-deficient (beclin1.sup.+/- and
atg5.sup.-/-) cells compared to wild-type cells. In order to
monitor the accumulation and clearance of p62, apoptosis-deficient
atg5.sup.+/+ and atg5.sup.-/- iBMK cells engineered to stably
express either EGFP or p62-EGFP were subjected to 3 days of
metabolic stress followed by 1 day recovery. As with myc-p62 (FIG.
1C), p62-EGFP-expressing atg5.sup.-/- iBMK cells displayed
persistent p62 aggregates that were induced during metabolic stress
and recovery while atg5.sup.+/+ iBMK cells were able to effectively
prevent their accumulation (FIGS. 6B and 6C). Consistent with
transient expression, stable p62-EGFP expression also activated the
DNA damage response (.gamma.-H2AX positive nuclei) during stress
and recovery, suggesting that accumulation of p62 was sufficient
for oxidative stress and DNA damage induction commonly observed
with autophagy-deficiency under stress (FIGS. 6B and 6C).
[0077] One of the mechanisms by which cells progress to aneuploidy
is by accumulation of supernumerary centrosomes leading to
multipolar spindles and cell division abnormalities, a
characteristic phenotype in stressed autophagy-deficient cells. To
determine whether p62-EGFP expression was sufficient to induce this
phenotype under metabolic stress, the frequency of centrosome and
cell division abnormalities in EGFP or p62-EGFP expressing
atg5.sup.+/+ and atg5.sup.-/- iBMK cells were analyzed. Unstressed,
EGFP-expressing atg5.sup.-/- iBMK cells showed a higher basal level
of supernumerary centrosomes (more than 2 per cell) (15.4%)
compared to atg5.sup.+/+ cells (1%), and p62-EGFP expression
increased the frequency of centrosome abnormalities in both
atg5.sup.-/- (25.8%) as well as in atg5.sup.+/+ (11.4%) iBMK cells.
Following metabolic stress (3 days) and recovery (1 day),
EGFP-expressing atg5.sup.-/- iBMK cells displayed increased
centrosomal abnormalities (45.3%) compared to the marginal increase
in atg5.sup.+/+ iBMK cells (4.6%) which was further increased by
p62-EGFP expression in the atg5.sup.-/- cells (78.5%) compared to
the atg5.sup.+/+ cells (15.4%), and caused multi-polar divisions at
markedly higher frequency in atg5.sup.-/- (65.1%) compared to
atg5.sup.+/+ (17.9%) iBMK cells. In contrast, control cells had a
more modest but higher frequency of multi-polar divisions in
atg5.sup.-/- (19.8%), compared to atg5.sup.+/+ (1.8%) cells where
p62 was progressively eliminated through autophagy. These
observations suggested that p62 accumulation was sufficient for ROS
and DNA damage response induction under metabolic stress and led to
the cell division abnormalities and genomic instability in
autophagy-defective cells. Indeed, RNAi-mediated knockdown of p62
during metabolic stress (3 days), reduced DNA damage induction in
autophagy-deficient beclin1.sup.+/- and atg5.sup.-/- iBMK cells,
further suggesting that the impairment of p62 elimination was the
cause of DNA damage response activation (FIGS. 6D-6F).
Example 7
p62 Promotes Tumorgenesis of Autophagy-Defective Cells
[0078] Since accumulation of p62 is a pronounced phenotype of
tissues and tumors from autophagy-deficient mice and some human
cancers, the possibility was investigated that it was capable of
promoting tumor growth. To this end, independently derived
apoptosis-deficient atg5.sup.+/+ and atg5.sup.-/- iBMK cell lines
expressing comparable levels of EGFP or p62-EGFP (FIG. 7A) were
assessed for their tumorigenic potential. p62-EGFP expression in
atg5.sup.+/+ iBMK cell lines did not substantially increase tumor
growth (FIG. 7B). In contrast, p62-EGFP expression in atg5.sup.-/-
cells dramatically increased the rate of tumor growth compared to
EGFP-expressing atg5.sup.-/- controls (FIGS. 7B-7D). By optical
imaging, tumors from p62-EGFP expressing atg5.sup.-/- cells were
uniformly fluorescent indicating that the p62 fusion protein was
expressed throughout the tumors (FIG. 7D). Fluorescence and
histological analysis of EGFP-expressing atg5.sup.-/- tumor
sections revealed diffuse cytoplasmic EGFP localization and
uniformly sized nuclei with occasional appearance of tumor giant
cells (FIG. 7E, H&E). In contrast, p62-EGFP-expressing
atg5.sup.-/- tumors showed dramatic p62 aggregate accumulation
(FIG. 7E, lower left panel), and numerous pleomorphic tumor cells
with heterochromatic, giant nuclei by H&E staining (FIG. 7E),
indicative of polyploidy and aneuploidy. Consistent with this,
persistent p62 accumulation in p62-EGFP expressing atg5.sup.-/-
tumors was also associated with markedly elevated DNA damage
response induction (.gamma.-H2AX staining) compared to
EGFP-expressing atg5.sup.-/- tumors (p<0.005, t-test) (FIG. 7E),
suggesting that inability to clear the p62 through autophagy
promoted tumor growth by elevating DNA damage and genomic
instability.
[0079] The results suggest that metabolic stress increases the
demand for protein and organelle turnover by autophagy, and
autophagy defects lead to induction and persistence of p62, which
is sufficient to elevate oxidative stress, DNA damage, genomic
instability and tumor progression. In autophagy-competent cells,
p62 is progressively degraded through autophagy, minimizing
oxidative stress and DNA damage (see model in FIG. 7F). These
results thus indicate a role for autophagy in tumor suppression by
damage mitigation under metabolic stress by eliminating p62.
Example 8
[0080] A cell-based screen utilizes apoptosis-defective
bax.sup.-/-bak.sup.-/- immortal baby mouse kidney epithelial (iBMK)
cells stably expressing the autophagosome marker EGFP-LC3
(D3-EGFP-LC3) (Degenhardt, K., Mathew, R., Beaudoin, B., Bray, K.,
Anderson, D., Chen, G., Mukherjee, C., Shi, Y., Gelinas, C., Fan,
Y., et al., (2006), Cancer Cell 10, 51-64). Autophagy promotes
tumor cell survival and restricts necrosis, inflammation, and
tumorigenesis. Under normal growth conditions EGFP-LC3 is diffusely
localized, and within 24 hours of metabolic stress in vitro and in
tumors in vivo, 80-90% of the cells display membrane translocation
and punctate localization of EGFPLC3 in autophagosomes. Deficiency
in Bax and Bak produces a profound defect in apoptosis, allowing
autophagy induction under stress without activating the apoptotic
response. Genetic inactivation of autophagy (loss of or down
regulation of Beclin1, Atg5 or Atg7) prevents relocalization of
EGFP-LC3, which remains diffuse. Although inhibition of autophagy
impairs survival to metabolic stress, the defect in apoptosis
permits survival sufficiently to clearly reveal autophagy
inhibition.
[0081] The apoptosis-defective iBMK cell line D3 stably expressing
the autophagosome marker EGFPLC3 is utilized to monitor EGFP-LC3
translocation to autophagosomes in response to metabolic stress.
Compounds and shRNAs that interfere with or promote autophagy
induction are identified and validated. The autophagy-defective
beclin1.sup.+/- EGFPLC3 iBMK cell line provides a negative control
for autophagosome formation in metabolic stress. HCQ treated
D3-EGFP-LC3 cells serve as a positive control for blockage of flux
through the autophagic pathway, resulting in increased
autophagosome accumulation.
[0082] This screen is used to identify inhibitors of EGFP-LC3
membrane translocation (resulting in no autophagosome formation) as
well as inhibitors of autophagosome trafficking, or lysosome fusion
and acidification (such as HCQ), the subsequent stages of the
autophagy pathway. The latter manifests as larger and more abundant
GFP-LC3 decorated autophagosomes throughout the cell due to
inhibition of flux through the pathway. Known autophagy inhibitors
are included in the assay as a control for blockage of autophagy
flux (FIGS. 12 and 8).
[0083] The shRNA screen is used to identify gene products that are
important for metabolic stress-mediated autophagy induction that
may be candidates for anti-cancer drug discovery targeting the
autophagy pathway. The small molecule screen is used to identify
compounds that are sufficient to disrupt the autophagic response to
metabolic stress. The targets of these compounds are determined in
subsequent validation studies.
Example 9
[0084] A cell-based screen utilizes autophagy defective
atg5.sup.-/- and beclin1.sup.+/- iBMK cells (Mathew et al., 2007b)
stably expressing EGFP-p62 (R. Mathew, C. M. Karp, B. Beaudoin, N.
Vuong, G. Chen, H.-Y. Chen, K. Bray, A. Reddy, G. Bhanot, C.
Gelinas, R. S. DiPaola, V. Karantza-Wadsworth, E. White, (2009),
Autophagy suppresses tumorigenesis through elimination of p62, Cell
137, 1062-1075). p62 accumulates and aggregates in response to
metabolic stress and requires autophagy for degradation. In shRNA
screens, genes are identified where inactivation compensates for
defective autophagy and restores p62 protein turnover (FIG. 9).
Similarly, small molecule screens identify compounds that
compensate for or restore functional autophagy by measuring the
elimination of p62 aggregates. Both the beclin1.sup.+/- and
atg5.sup.-/- autophagy-defective cells are screened in this assay,
as fundamentally different mechanisms may govern compensation for a
haploinsufficient and a null defect, respectively.
[0085] The autophagy-defective atg5.sup.-/- and beclin1.sup.+/-
iBMK cell lines stably expressing EGFP-p62 accumulate
p62-containing protein aggregates under stress, which fail to be
cleared following recovery. Those shRNAs or small molecules that
facilitate p62 aggregate clearance, compensating for defective
autophagy, are identified and subjected to future validation. The
autophagy wild type atg5.sup.+/+ iBMK cell line stably expressing
EGFP-p62 that effectively clears p62 aggregates following stress is
used as a positive control. mTOR inhibitors and an mTOR-independent
stimulator of autophagy, trehalose, are tested as a potential
positive control for p62 elimination in atg5.sup.-/- or
beclin1.sup.+/- cells.
[0086] While not wishing to be bound by any theory or theories of
operation, evidence suggests that promoting autophagy and the
clearance of misfolded proteins can greatly mitigate disease
progression. The cell-based screens described herein may be used to
screen for genes where down regulated expression suppresses
metabolic stress-mediated p62 accumulation in autophagy-defective
atg5.sup.-/- and beclin1.sup.+/- cells. These genes are expected to
be therapeutic targets for small molecule inhibitors for prevention
and/or treatment of diseases including, but not limited to,
neurodegeneration, liver disease and cancer. Additionally, chemical
libraries may be screened for compounds that can restore autophagy
and result in p62 aggregate elimination in well-characterized
autophagy-deficient cell lines.
[0087] The terms and expressions which have been employed are used
as terms of descriptions and not of limitation, and there is no
intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention. Thus, it should be
understood that although the present invention has been illustrated
by specific embodiments and optional features, modification and/or
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this
invention.
[0088] In addition, where features or aspects of the invention are
described in terms of Markush group or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
[0089] The disclosures of each patent, patent application and
publication cited or described in this document are hereby
incorporated herein by reference, in their entireties.
* * * * *