Molecular Biology of the Cell
Vol. 19, 797–806, March 2008
Induction of Autophagy during Extracellular Matrix
Detachment Promotes Cell Survival
Christopher Fung,* Rebecca Lock,*†Sizhen Gao,‡Eduardo Salas,*
and Jayanta Debnath*†
*Department of Pathology and†Biomedical Sciences Graduate Program, University of California San
Francisco, San Francisco, CA 94143; and‡Department of Cell Biology, Harvard Medical School, Boston,
Submitted October 30, 2007; Revised December 1, 2007; Accepted December 12, 2007
Monitoring Editor: Donald Newmeyer
Autophagy has been proposed to promote cell death during lumen formation in three-dimensional mammary epithelial
acini because numerous autophagic vacuoles are observed in the dying central cells during morphogenesis. Because these
central cells die due to extracellular matrix (ECM) deprivation (anoikis), we have directly interrogated how matrix
detachment regulates autophagy. Detachment induces autophagy in both nontumorigenic epithelial lines and in primary
epithelial cells. RNA interference-mediated depletion of autophagy regulators (ATGs) inhibits detachment-induced
autophagy, enhances apoptosis, and reduces clonogenic recovery after anoikis. Remarkably, matrix-detached cells still
exhibit autophagy when apoptosis is blocked by Bcl-2 overexpression, and ATG depletion reduces the clonogenic survival
of Bcl-2–expressing cells after detachment. Finally, stable reduction of ATG5 or ATG7 in MCF-10A acini enhances
luminal apoptosis during morphogenesis and fails to elicit long-term luminal filling, even when combined with apoptotic
inhibition mediated by Bcl-2 overexpression. Thus, autophagy promotes epithelial cell survival during anoikis, including
detached cells harboring antiapoptotic lesions.
Macroautophagy (hereafter called autophagy) is an evolu-
tionarily conserved lysosomal process where a cell degrades
its own cytoplasmic contents (Levine and Klionsky, 2004).
Accumulating evidence indicates that the exact role of au-
tophagy in cell survival versus death is both stimulus and
context dependent (Debnath et al., 2005; Levine and Yuan,
2005). Indeed, autophagy is well recognized as a survival
mechanism during nutrient limitation; through the bulk
degradation of cytoplasmic material, autophagy generates
both nutrients and energy in starving cells (Levine and
Klionsky, 2004). Accordingly, during nutrient starvation,
inhibition of autophagy promotes apoptosis (Boya et al.,
2005). In contrast, excessive autophagy has been proposed to
mediate autophagic or type 2 programmed cell death (PCD).
For example, L929 fibrosarcoma cells die in a caspase-inde-
pendent manner involving autophagy; ATG genes are re-
quired for this death process (Yu et al., 2004). In this model,
caspase inhibition induces the selective autophagic degra-
dation of catalase, a major reactive oxygen species (ROS)
scavenger, and the resulting ROS accumulation promotes
type 2 PCD (Yu et al., 2006). Type 2 PCD has also been
described during development, most notably, during the
destruction of larval salivary glands in Drosophila (Lee and
Baehrecke, 2001; Martin and Baehrecke, 2004). Presumably,
the level of self-degradation becomes incompatible with life,
leading to cellular demise.
Both autophagy and apoptosis are observed during lumen
formation in three-dimensional (3D) epithelial cultures in
vitro. When cultured on reconstituted basement membrane,
MCF-10A cells, a nontransformed human mammary epithe-
lial cell line, form spherical structures (termed “acini”) in
which a layer of polarized epithelial cells surrounds a hol-
low lumen, resembling glandular epithelium in vivo (Deb-
nath et al., 2003). The detailed analysis of 3D morphogenesis
reveals the presence of two populations in developing acini:
an “outer” cell layer directly attached to the extracellular
matrix (ECM), and a centrally located subset of “inner” cells
lacking ECM contact. Lumen formation involves the selec-
tive apoptosis of these central cells; caspase-3 cleavage is
observed in dying cells as the lumen hollows (Debnath et al.,
2002). However, inhibiting apoptosis by the ectopic expres-
sion of either Bcl-2 or Bcl-xLonly delays lumen clearance for
a few days. Ultimately, these acini do form hollow lumen.
Electron microscopic analysis reveals that numerous auto-
phagic vacuoles are present in the central cells of developing
acini (Debnath et al., 2002; Underwood et al., 2006). Notably,
the central cells in Bcl-2–expressing structures also exhibit
extensive autophagy (Debnath et al., 2002).
Based on these initial results, autophagy has been proposed
to promote luminal clearance by autophagic death (Debnath et
al., 2002; Mills et al., 2004). Notably, follow-up studies indicate
that a tumor necrosis factor family ligand, Tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) induces au-
tophagy in epithelial cells and that TRAIL inhibition promotes
luminal filling when combined with Bcl-xL–mediated inhibi-
This article was published online ahead of print in MBC in Press
on December 19, 2007.
Abbreviations used: ATG, autophagy gene; AMPK, AMP-activated
protein kinase; ECM, extracellular matrix; EGFR, epidermal growth
factor receptor; eIF2?, eukaryotic initiation factor 2?; GFP-LC3,
green fluorescent protein fused to light chain 3; LC3, light chain 3;
PE, phosphotidylethanolamine; 3D, three-dimensional.
© 2008 by The American Society for Cell Biology797
tion of apoptosis (Debnath et al., 2002; Mills et al., 2004). Al-
though these results suggest that both apoptosis and autoph-
agy are required for luminal cell death and clearance, these
studies are correlative (Debnath et al., 2005). Importantly, epi-
thelial cells critically depend on integrin-mediated cell adhe-
sion to ECM for proper growth and survival; upon detach-
ment, epithelial cells undergo apoptotic cell death, termed
anoikis (Frisch and Francis, 1994). Thus, one can alternatively
hypothesize that autophagy is induced during anoikis as a
survival strategy to mitigate the stresses of ECM detachment.
Because protection from anoikis is thought to promote tumor
cell survival in vivo and to contribute to luminal filling in
glandular structures, we interrogated whether autophagy is
induced in epithelial cells due to ECM detachment. We also
tested whether autophagy regulators (ATGs) promote epithe-
lial cell survival, versus type 2 autophagic cell death, during
anoikis and 3D lumen formation.
MATERIALS AND METHODS
MCF-10A cells were cultured as described previously (Debnath et al., 2003).
Primary human mammary epithelial cells (1°HMECs) were obtained from
Cambrex (East Rutherford, NJ) and cultured in Mammary Epithelial Cell
Medium (MEGM) (Cambrex). For epidermal growth factor (EGF) withdrawal
studies, EGF was omitted as an exogenous supplement from either MCF-10A
or MEGM growth media. Madin-Darby canine kidney (MDCK)-2 cells (gift
from Dr. K. Matlin, University of Cincinnati, Cincinnati, OH) and both
wild-type and ATG5?/? simian virus 40-immortalized mouse embryonic
fibroblasts (gift from Dr. N. Mizushima, Tokyo Medical and Dental Univer-
sity, Tokyo, Japan) were grown in DMEM (Invitrogen, Carlsbad, CA) supple-
mented with 10% fetal bovine serum, penicillin, and streptomycin.
Antibodies and Chemicals
A peptide corresponding to the N terminus common to human, mouse, and
rat MAP1LC3 was used to create ?-LC3 rabbit polyclonal antibody; the whole
sera was used at 1:500–1:1000 for immunoblotting. In mammary cells, we
noticed that LC3-I is not efficiently detected with this antibody when low
amounts of protein lysates are analyzed. (e.g., see Figure 3B). Other antibodies
used included the following: ?-ATG12 (Zymed Laboratories, South San Fran-
cisco, CA); ?-ATG5 (gift from N. Mizushima, Tokyo Medical and Dental
University); ?-ATG7 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); ?-Be-
clin (ATG6) (BD Biosciences, San Jose, CA); ?-Bim (Prosci, Poway, CA);
?-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA); ?-catalase
(Calbiochem, San Diego, CA); ?-enhanced epidermal growth factor receptor
(EGFR) (Cell Signaling Technology); ?-laminin 5 (Chemicon International,
Temecula, CA); ?-?-tubulin (Sigma-Aldrich, St. Louis, MO); ?-phospho-
AMP-activated protein kinase (AMPK), ?-AMPK?, ?-P-eukaryotic initiation
factor 2? (eIF2?), and eIF2? (Cell Signaling Technology); ?-phopho-ERK1/2
(BioSource International. Camarillo, CA); and ?-extracellular signal-regulated
kinase (ERK)1/2 (Zymed Laboratories). For function-blocking studies, ?-?1 in-
tegrin subunit (A2B2) was from C. Damsky (University of California San Fran-
cisco, San Francisco, CA) and ?-E-cadherin (DECMA) and the rat immunoglob-
ulin (Ig)G isotype control were from Sigma-Aldrich. Chemicals utilized included
poly(2-hydroxyethyl methacrylate) (poly-HEMA), 3-methyadenine, bafilomycin
A, chloroquine, E64d, and pepstatin A (all from Sigma-Aldrich).
Generation of Stable Lines
The following retroviral vectors for stable gene expression have been de-
scribed previously: pBABEpuro-Bcl-2, pBABEneo-Bcl-2, pBABEhygro-Bcl-xL,
and pLPCX-EGFR (Debnath et al., 2002; Reginato et al., 2003). Rat green
fluorescent protein fused to the mammalian ATG8 orthologue, microtubule-
associated protein light chain 3 (GFP-LC3), was subcloned from pEGFPC1-
LC3, a gift from T Yoshimori (National Institute of Genetics, Mishima, Japan),
into the SnaB1 and Sal1 sites of pBABEpuro (Kabeya et al., 2000). Vesicular
stomatitis virus G protein-pseudotyped retroviruses were generated, and
MCF-10A lines were infected and selected as described previously (Debnath
et al., 2003).
Pooled small interfering RNA (siRNA) oligonucleotides (SMARTpool) against
ATG5, ATG6 (Beclin 1), or ATG7 were purchased from Dharmacon RNA Tech-
nologies (Lafayette, CO). For siRNA transfection, cells were seeded at 100,000/
well in six-well tissue dishes, and they were transfected with 50–100 nM of the
pooled oligonucleotide mixture by using Oligofectamine (Invitrogen) following
manufacturer’s protocols. The transfection media were removed, and cells were
allowed to recover in complete growth media for 36–48 h before use in experi-
ments. The sense sequences of the individual duplexes directed against ATG5, 6,
and 7 are as follows: ATG5 (NM_004849): GGAAUAUCCUGCAGAAGAAUU,
CAUCUGAGCUACCCGGAUAUU, GACAAGAAGACAUUAGUGAUU, and
CAAUUGGUUUGCUAUUUGAUU; BECN1(ATG6) (NM_003766): CUAAG-
GAGCUGCCGUUAUAUU, GGAUGACAGUGAACAGUUAUU, UAAGAUG-
GGUCUGAAAUUUUU, and GCCAACAGCUUCACUCUGAUU; and ATG7
(NM_006395): CCAAAGUUCUUGAUCAAUAUU, GAUCAAAGGUUUUCA-
CUAAUU, GAAGAUAACAAUUGGUGUAUU, and CAACAUCCCUGGUU-
DNA oligonucleotides encoding short hairpin RNA (shRNA) sequences
against ATG5 were chemically synthesized (Dharmacon RNA Technologies),
annealed, and subcloned into BglII and XhoI sites of the pSRpuro retroviral
vector to express shRNAs under the control of the H1 promoter. The target
sequence of two small hairpin RNAs against ATG5 are as follows: shATG5hp1,
GGATGAGATAACTGAAAGG; and shATG5hp2, GGCATTATCCAATTG-
Lentiviral particles expressing small hairpin RNAs against ATG7 were
purchased from Sigma-Aldrich (Mission shRNA). The target sequence of the
two hairpins used were shATG7hp1 (NM_006395; TRCN0000007584), GCCT-
GCTGAGGAGCTCTCCAT; and shATG7hp2 (TRCN0000007587), CCCAGC-
Substratum Detachment Assays
Tissue culture plates were coated with 6 mg/ml poly-HEMA in 95% ethanol,
and then they were incubated at 37°C for several days until dry. Cells were
plated on poly-HEMA–coated plates at a density of 100,000–250,000 cells/
well in their appropriate complete growth medium. To analyze LC3-II turn-
over, the lysosomal inhibitors E64d and pepstatin A were added directly to
the culture media at 10 ?g/ml at 2–4 h before harvest. To quantify apoptosis,
cells were suspended or attached for 24 h, collected, fixed, and stained with
cleaved caspase-3 antibody by using protocols described previously (Debnath
et al., 2003). To enumerate the percentage of cells positive for cleaved
caspase-3, 200–300 cells were counted for each condition, and each experi-
ment was repeated at least five times. For electron microscopy of detached
cells, samples were processed at the Harvard Medical School Electron Mi-
croscopy core facility by using protocols described previously, and they were
imaged using a JEOL-60kV microscope (Debnath et al., 2002; Mills et al., 2004).
Analysis of Punctate GFP-LC3
MCF-10A or 1°HMECs stably expressing GFP-LC3 were grown overnight
attached on 2% Matrigel-coated coverslips before EGF withdrawal or starva-
tion with Hank’s balanced salt solution (HBSS) for the indicated times, or
grown suspended for the indicated times on poly-HEMA–coated plates. Cells
were fixed with 2% paraformaldehyde, washed several times with phosphate-
buffered saline (PBS), mounted using Immunomount (Thermo Electron,
Waltham, MA), and analyzed at 20°C by widefield immunofluorescent micros-
copy by using the 63? (1.4 numerical aperture [NA]) or 100? (1.3 NA) objectives
of an Axiovert 200 microscope (Carl Zeiss, Thornwood, NY) equipped with a
Spot RT camera (Diagnostic Instruments, Sterling Heights, MI) and mercury
lamp; images were acquired using MetaMorph (version 6.0) software (GE
Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).
Clonogenic Replating Assays
Cells (100,000) were grown attached or suspended on poly-HEMA–coated
dishes for 48 h, collected, and treated with 0.25% trypsin-EDTA at 37°C for 15
min to generate single cell suspensions. Cells were counted and replated in
complete growth media at 100 cells/well onto 12-well tissue culture plates.
Colonies were grown out for 5 d, fixed with 2% paraformaldehyde, and
stained with 0.2% crystal violet in PBS. The number of colonies was enumer-
ated, and replating efficiency was calculated as the number of colonies grow-
ing out divided by the original number of cells plated. For each experiment,
four to six replicates were performed for each condition tested, and each
experiment was repeated three to five times.
3D Morphogenesis Assays
3D assays were carried out and processed for ethidium bromide (EtBr)
staining or confocal microscopy as described previously (Debnath et al., 2003).
Where indicated, 10 mM 3-methyladenine, 20 nM bafilomycin A, or 20 ?M
chloroquine was added on day 6.
Attached or suspended cells were lysed in radioimmunoprecipitation assay
(1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 25 mM Tris, pH 7.6, 150
mM NaCl, 10 mM NaF, 10 mM ?-glycerophosphate, 1 mM Na3VO3, and 10
nM calyculin A) plus protease inhibitors. Lysates were cleared by centrifu-
gation for 15 min at 4°C, boiled in SDS sample buffer, resolved using
SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to polyvi-
nylidene difluoride membrane. The membranes were blocked in PBS ? 0.1%
Tween 20 with 5% nonfat dry milk, incubated with the primary antibodies
indicated overnight at 4°C, washed, incubated with horseradish peroxidase-
C. Fung et al.
Molecular Biology of the Cell 798
conjugated secondary antibodies, and analyzed by enhanced chemilumines-
Immunofluorescence and Confocal Microscopy
Widefield immunofluorescence imaging was performed on an Axiovert 200
microscope (Carl Zeiss) equipped with a mercury lamp. Images were ac-
quired at 20°C with a 20? (0.4 NA) objective with a Spot RT charge-coupled
device (CCD) camera (Diagnostic Instruments) and MetaMorph (version 6.0)
software. Confocal imaging was performed at 20°C, by using an Axiovert 200
(Carl Zeiss) equipped with a Yokogawa CSU-10 spinning disk, an argon laser
(488 line), and two solid-state diode lasers (405 and 546 lines). Images were
acquired using a 40? (1.3 NA) objective with an Andor iXON CCD camera
and MetaMorph (version7.0) software. Images were color-combined in Meta-
Morph (version 6.0).
Substratum Detachment Induces Autophagy in Epithelial
Cells and Fibroblasts
To monitor autophagosome formation, we created MCF-10A
cells and 1°HMECs that stably express GFP-LC3. During
autophagy, LC3 is modified with the lipid phospatidyleth-
anolamine (PE) through an ubiquitin-like conjugation pro-
cess, upon which it specifically relocates to early autopha-
gosomes; accordingly, the relocation of GFP-LC3 to easily
visualized “puncta” has emerged as a powerful technique to
monitor autophagosome formation (Kabeya et al., 2000). Be-
cause transient GFP-LC3 transfection can produce overex-
pression artifacts, we developed retroviral vectors (pBABE)
encoding GFP-LC3, and we generated stable pools ectopi-
cally expressing these fusions (Kuma et al., 2007). In these
cells, we confirmed that GFP-LC3 relocates to autophago-
somes during HBSS-induced nutrient starvation (Figure 1A).
Integrin-mediated cell adhesion to ECM is critical for
proper growth and survival (Meredith et al., 1993; Frisch and
Francis, 1994). Thus, we hypothesized that the lack of ECM
contact would induce autophagy in mammary epithelial
cells during anoikis, a type of apoptotic cell death that
epithelial cells undergo when detached from an underlying
substratum for extended periods (Frisch and Francis, 1994).
To induce anoikis, we incubated cells on poly-HEMA–
coated tissue culture dishes to prevent cell attachment. Al-
though epithelial cells form clusters in these conditions due
to increased cell–cell contact, they ultimately undergo apo-
ptosis due to the lack of ECM contact (Frisch, 1999; Reginato
et al., 2003). We found that substratum detachment strongly
induced GFP-LC3 puncta in both MCF-10A cells and
1°HMECs (Figure 1A). In addition, using electron micros-
copy, we corroborated the presence of autophagic vacuoles;
double membrane vacuoles containing cytoplasmic contents
were observed in detached cells (Figure 1B).
In contrast to cells grown as monolayers (Supplemental
Figure S1), we were unable to reliably quantify the number
of GFP-LC3 puncta per cell by using image analysis soft-
ware, due to the clustering of cells during anoikis. As a
result, to measure autophagy during anoikis, we examined
the PE-lipid conjugation of endogenous LC3 in cell lysates
from detached cells; the PE-modification of LC3 results in a
faster migrating isoform (called LC3-II) that can be detected
by immunoblotting (Kabeya et al., 2000). Increased PE-mod-
ified LC3-II was observed in MCF-10A cells after 24 h of
detachment (Figure 2A). Because LC3-II is subject to lysoso-
mal degradation during autophagy, increased LC3-II levels
do not always accurately reflect full-fledged autophagic deg-
radation (Tanida et al., 2005). To more precisely assess au-
tophagic flux, we assessed LC3-II levels in both attached
controls and detached cells treated with lysosomal cathepsin
inhibitors E64d and pepstatin A. These experiments con-
firmed increased LC3-II turnover during ECM detachment
(Figure 2B, E64d/pepA ? lanes). Interestingly, in attached
1°HMECs, we found high baseline levels of LC3-II that were
reduced upon detachment (Figure 2C, E64d/pepA ? lanes).
Nonetheless, we once again observed increased LC3-II in the
presence of these inhibitors (Figure 2C, E64d/pepA ?
lanes). Altogether, these experiments confirmed increased
LC3-II turnover, consistent with increased autophagic deg-
radation in MCF-10A cells and 1°HMECs during substratum
Furthermore, we examined whether autophagy was in-
duced in other cells during matrix detachment. We observed
tion in human mammary epithelial cells. (A) MCF-10A cells or
1°HMECs expressing GFP-LC3 were grown attached in full growth
media for 48 h (control, left column), starved with Hank’s balanced
salt solution for 3 h (HBSS, center), or grown detached on poly-
HEMA–coated plates in complete growth media for 48 h (suspend,
right). Bars, 20 ?m. (B) MCF-10A cells grown detached for 24 h were
analyzed by transmission electron microscopy. The boxed area in
upper panel is enlarged in lower panel. Bars, 200 nm.
Substratum detachment induces autophagosome forma-
fibroblasts. (A) Lysates from MCF-10A cells grown attached (A) for
48 h or suspended for the indicated times were immunoblotted with
?-LC3 and ?-tubulin. (B and C) MCF-10A and 1°HMEC lysates
grown attached (A) or suspended for the indicated times were
subject to ?-LC3 and ?-tubulin immunoblotting. When indicated by
E/P?, E64d and pepstatin A (10 ?g/ml each) were added directly to
the culture 4 h before lysis. (D) Cell lysates collected from MDCK2 cells
grown attached for 48 h (A) or suspended for the indicated times in
A (10 ?g/ml each) were added directly to the culture 4 h before lysis.
(E) Wild-type or ATG5?/? MEFs were grown attached for 48 h or
detached in complete media for the indicated times. All lysates were
subject to immunoblotting with ?-LC3 and ?-tubulin antibodies.
Detachment-induced autophagy in epithelial cells and
Autophagy during Anoikis
Vol. 19, March 2008799
increased LC3-II turnover during ECM detachment in
MDCK2 cells (Figure 2D), a nontumorigenic epithelial cell
line susceptible to anoikis (Rytomaa et al., 2000). Finally, we
found increased LC3-II formation during the detachment of
mouse embryonic fibroblasts (MEFs), which do not undergo
apoptosis during matrix detachment, but critically depend
on integrin-based signals for proper cell growth and prolif-
eration (Miranti and Brugge, 2002). LC3-II conversion was
potently inhibited in autophagy deficient ATG5?/? MEFs,
although increased unmodified LC3 (LC3-I) levels were ob-
served (Figure 2E) (Kuma et al., 2004). Thus, autophagy is a
general response to substratum detachment in various epi-
thelial cells and in mouse fibroblasts.
We next sought to clarify whether the autophagy induced
by substratum detachment could more directly result from
reduced ECM or integrin-mediated signals. When exoge-
nous laminin-rich reconstituted basement membrane (2%)
was added to suspended cells, LC3-II formation decreased
(Supplemental Figure S2A). We speculate that these rescue
experiments did not completely inhibit autophagy because
exogenous basement membrane addition fails to restore
the mechanical forces critical for proper integrin function
(Katsumi et al., 2004). To further interrogate the role of
integrin receptor engagement, we examined autophagy in
cells incubated with function-blocking antibodies directed
against the ?1 integrin subunit, a critical regulator of anoikis in
ade of ?1 integrin (using A2B2) increased LC3-II formation
(Supplemental Figure S2B). In contrast, we did not observe the
induction of LC3-II upon inhibiting E-cadherin–mediated cell–
cell adhesion by using a blocking antibody (DECMA) (Supple-
mental Figure S2B). Thus, a laminin-rich matrix or reduced ?1
integrin function can modulate autophagosome formation in
detached epithelial cells.
ATG Depletion Enhances Apoptosis in Detached Cells
Autophagy is tightly regulated by a limited number of
highly conserved molecules called ATGs (Klionsky et al.,
2003). Because ATG depletion inhibits autophagy, we used
siRNA oligonucleotide pools targeting human ATG5, 6, and
7 to reduce autophagy during matrix detachment. We con-
firmed siRNA-mediated reduction of endogenous ATG5,
ATG6 (Beclin1), and ATG7 in MCF-10A cells by immuno-
blotting (Figure 3A), which all produced significant de-
creases in LC3-II during anoikis (Figure 3B). In parallel, we
quantified how ATG reduction affects GFP-LC3 puncta for-
mation during HBSS starvation; cells with reduced ATG5
exhibited an 80–90% reduction in GFP-LC3 dots per cell,
whereas cells with reduced ATG6 and 7 exhibited milder
inhibition (Supplemental Figure S1A). Overall, these results
indicated that silencing of multiple ATGs results in reduced
autophagy. Cells with reduced ATGs were subject to matrix
detachment for 24 h, and the levels of apoptosis were mea-
sured by enumerating the percent of cells within a culture
with positive staining for cleaved caspase-3 (Figure 3C). In
cells with reduced ATGs, we observed a 1.5- to 2.0-fold
increase in cleaved caspase-3 in suspended cells; in contrast,
no differences in apoptosis were observed in attached con-
trols (Figure 3D). Notably, these proapoptotic effects were
observed with the silencing of multiple independent ATGs,
arguing against nonspecific effects due to any individual
ATG knockdown. Induction of the proapoptotic BH3-family
protein Bim critically mediates apoptosis in detached mam-
mary epithelial cells (Reginato et al., 2003). As a result, we
examined whether ATG depletion amplified Bim induction
during anoikis; however, we did not observe changes in Bim
protein levels in cells with reduced ATGs versus controls
(Supplemental Figure S3).
Finally, to more directly interrogate how autophagy regu-
lates cell viability after anoikis, we assayed clonogenic poten-
tial after ECM detachment. After 48 h of matrix detachment,
single cell suspensions were generated from control and ATG-
depleted cells and replated to assess clonogenic recovery and
colony formation. In cells subjected to ECM detachment, we
found decreased colony formation in ATG-depleted cultures
versus controls; in contrast, reduced clonogenicity was not
in ECM-detached cells. (A) MCF-10A cells
transfected with pooled nontargeting control
(siControl) or siRNA oligonucleotides against
ATG5, ATG6 (Beclin 1), or ATG7 were lysed
and immunoblotted with ?-ATG12 to detect
the ATG5:12 complex, ?-Beclin, ?-ATG7, and
?-tubulin. (B) Cells transfected with the indi-
cated siRNAs were grown attached (A) or
suspended (S) for 24 h, lysed, and subjected to
?-LC3 and ?-tubulin immunoblotting. LC3-I
detection was minimal in this experiment.
(C) Confocal images of MCF-10A cells trans-
fected with the indicated siRNAs, suspended
for 24 h, fixed, and immunostained with
?-cleaved caspase-3 antibody (green). Nuclei
were stained with 4,6-diamidino-2-phenylin-
dole (DAPI) (blue). Bars, 20 ?m. (D) Percent-
age of cleaved caspase-3–positive cells in
MCF-10A cells transfected with the indicated
siRNAs and grown attached (white) or sus-
pended (black) for 24 h. Results are the
mean ? SEM of five or more independent
experiments; in each experiment, 200–300
cells were analyzed. (E) Clonogenic replating
efficiency of cells transfected with the indi-
cated siRNAs after 48-h attachment (white) or
ATG depletion enhances apoptosis
suspension (black). Replating efficiency was calculated as the number of colonies formed divided by the number of cells originally replated.
Results are the mean ? SEM from three to five independent experiments.
C. Fung et al.
Molecular Biology of the Cell800
observed in attached cells with reduced ATGs (Figure 3E).
Thus, similar to cells undergoing nutrient starvation, the inhi-
bition of autophagy promotes apoptosis and decreases the
survival of cells deprived of ECM contact (Boya et al., 2005).
Detachment-induced Autophagy Promotes the Survival of
Cells Expressing Bcl-2
Our previous studies of lumen formation in Bcl-2–overex-
pressing acini indicate that apoptosis inhibition does not
affect autophagy induction in cells occupying the lumen
(Debnath et al., 2002; Mills et al., 2004). Accumulating evi-
dence indicates that depending on cell type, context, or both,
Bcl-2 inhibits autophagy (Pattingre et al., 2005), simulates
autophagy and ATG-dependent cell death (Shimizu et al.,
2004), or permits autophagy to develop by inhibiting apo-
ptosis (Degenhardt et al., 2006). To distinguish among these
possibilities, we examined the induction of autophagy dur-
ing matrix detachment in cells expressing antiapoptotic
Bcl-2 family proteins. We observed no significant changes in
GFP-LC3 puncta development between control versus Bcl-
xL–expressing cells (Figure 4A). Similarly, by immunoblot
analysis, we found no significant differences in PE-modified
LC3-II within Bcl-2–expressing cells during anoikis (Figure
4B). These results indicate that antiapoptotic Bcl-2 proteins
do not inhibit detachment-induced autophagy in mammary
When caspases are pharmacologically inhibited, catalase,
a major reactive oxygen species (ROS) scavenger is selec-
tively degraded by autophagy, which has been demon-
strated to mediate type 2 autophagic death (Yu et al., 2006).
Thus, to investigate the possibility that autophagy regulated
type 2 cell death during ECM detachment, we examined the
protein levels of catalase during anoikis. However, we ob-
served slightly increased, rather than decreased, catalase
protein levels in both wild-type and Bcl-2–expressing cells
(Figure 4C). Thus, we conclude that catalase depletion does
not mediate death during ECM detachment, even when
apoptosis is inhibited by Bcl-2. Interestingly, we observed
increased catalase protein levels in ATG knockdown cells
undergoing anoikis compared with controls (Figure 4D).
Although these results support that autophagy directly me-
diates catalase degradation during anoikis, increased cata-
lase may arise as a secondary consequence of increased
reactive oxygen species levels in detached ATG knockdown
Growing evidence indicates that autophagy promotes the
viability of cells unable to undergo apoptosis. When lym-
phocytes doubly deficient for Bax and Bak are deprived of a
critical growth factor, interleukin-3, the inhibition of auto-
phagy elicits a rapid cell death associated with reduced ATP
levels (Lum et al., 2005). Similarly, in cells unable to undergo
apoptosis due to Bcl-2 or Bcl-xL expression, Beclin (ATG6)
depletion impairs the induction of autophagy during ischemia
and reduces cell viability (Degenhardt et al., 2006). Based on
these previous studies, we hypothesized that autophagy may
contribute to the survival of Bcl-2–expressing cells during ma-
trix detachment. We measured the clonogenic viability of Bcl-
2–expressing cells after 48 h of detachment. Similar to our
results in wild-type cells, the knockdown of ATG5, 6, or 7
resulted in decreased colony formation when compared with
Bcl-2–expressing controls (Figure 4E). As before, no differences
in clonogenicity were observed in attached cells. Thus, detach-
ment-induced autophagy promotes the viability of cells over-
expressing the antiapoptotic protein Bcl-2.
Autophagy Is Induced during Epidermal Growth Factor
Substratum attachment and integrin-mediated cell adhesion
are essential for the proper activation of growth factor re-
ceptor pathways (Miranti and Brugge, 2002). Specifically,
EGFR, a growth factor receptor important for epithelial cell
survival and proliferation, is significantly reduced in a va-
riety of epithelial cells during anoikis (Reginato et al., 2003).
When we examined EGFR levels in Bcl-2–expressing cells,
we found that these cells exhibited EGFR down-regulation
similar to wild-type controls (Figure 5A). Thus, we reasoned
that even though Bcl-2–expressing cells were protected from
apoptosis, they would still exhibit other biological conse-
quences associated with EGFR down-regulation during
ECM detachment. Moreover, we postulated that detach-
ment-induced autophagy in both wild type and Bcl-2–ex-
pressing cells was at least partially due to reduced EGFR
expression and pathway activation.
To test this hypothesis, we first assessed whether EGF
depletion was sufficient to induce autophagy in mammary
cells. (A) Punctate GFP-LC3 in detached MCF-10A cells stably ex-
pressing GFP-LC3 or coexpressing GFP-LC3 ? Bcl-xLsuspended for
24 h. Bar, 20 ?m. (B) Control (BABE) or Bcl-2–expressing cells were
grown attached (A) for 48 h or suspended for the indicated times,
lysed, and subject to immunoblotting with ?-LC3 and ?-tubulin
antibodies. (C) Wild-type and Bcl-2–expressing cells were grown
attached (A) or suspended for the indicated times, lysed, and subject
to immunoblotting with ?-catalase and ?-tubulin antibodies. (D)
Cells transfected with the indicated siRNAs were grown attached
(A) or suspended for the indicated times, lysed, and subject to
immunoblotting with ?-catalase and ?-tubulin antibodies. (E) Clo-
nogenic replating efficiency of Bcl-2–expressing cells transfected
with the indicated siRNAs after 48 h attachment (white) or suspen-
sion (black). Replating efficiency was calculated as described previ-
ously. Results are the mean ? SEM from three to five independent
Detachment-induced autophagy in Bcl-2 expressing
Autophagy during Anoikis
Vol. 19, March 2008801
cells when grown in attached conditions. Indeed, we discov-
ered that EGF withdrawal induced autophagy in both MCF-
10A cells and 1°HMECs (Figure 5B). Importantly, autophagy
during EGF withdrawal ensued despite the presence of
other growth factors, including insulin, hydrocortisone, and
serum. Notably, we observed that autophagy was a revers-
ible process; upon EGF readdition, GFP-LC3 puncta disap-
peared within 12 h (Figure 6B). After 24 h of EGF depletion,
a fourfold increase in GFP-LC3 puncta was observed in
MCF-10A cells. Moreover, we did not detect reduced levels
of punctate GFP-LC3 in cells ectopically expressing Bcl-2, in
contrast to ATG depletion during EGF withdrawal (Figure
5C and Supplemental Figure S1). Furthermore, during EGF
withdrawal in both control and Bcl-2–expressing cells, we
observed increased LC3-II turnover in the presence of E64d
and pepstatin A (Figure 5D). Overall, these results indicated
that EGF withdrawal was sufficient to induce autophagy in
both wild-type and Bcl-2–expressing mammary epithelial
We then tested whether the ectopic overexpression of
EGFR was sufficient to inhibit detachment-induced autoph-
agy. Surprisingly, we found that stable EGFR overexpres-
sion did not significantly inhibit LC3-II formation and turn-
over during matrix detachment; in addition, we observed
the robust punctate GFP-LC3 in suspended EGFR-overex-
pressing cells (Figure 6, A and B). Based on this result, we
hypothesized that although the loss of EGFR driven signals
may contribute to autophagy, additional EGFR-independent
pathways also positively regulated autophagy during anoikis.
Thus, we probed whether specific survival and stress sig-
naling pathways were regulated by EGFR during anoikis,
whereas others were not. Indeed, we confirmed that EGFR-
overexpressing cells exhibited potent and sustained activa-
tion of certain survival pathways during matrix detachment,
most notably, the ERK/mitogen-activated protein kinase
pathway, previously demonstrated to inhibit apoptosis (Fig-
ure 6C; Reginato et al., 2003). In contrast, we uncovered that
several other stress response pathways were not inhibited
by EGFR overexpression. First, during anoikis, we observed
high levels of activation of the energy sensor AMPK in both
control and EGFR-overexpressing cells (Figure 6D). Increas-
ing evidence indicates that AMPK positively regulates au-
agy. (A) Lysates from pBABE control and Bcl-
2–expressing MCF-10A cells grown attached
(A) or suspended for the indicated times were
subject to immunoblotting with ?-EGFR and
?-tubulin. (B) GFP-LC3–expressing MCF-10A
cells or 1°HMECs were grown in media lack-
ing EGF for the indicated times. Twelve hours
of EGF readdition reverses LC3 puncta forma-
tion. (C) GFP-LC3 puncta in pBABE control
and Bcl-2–expressing cells after 24 h of EGF
withdrawal. Bar, 20 ?m. Bottom, quantifica-
tion of GFP-LC3 dots per cell in control and
Bcl-2 cells grown in complete growth media
(black), HBSS starved for 5 h (light gray), or
EGF withdrawn for 24 h (dark gray). Results
are the mean ? SEM enumerated from 40 to
60 individual cells using MetaMorph (GE
Healthcare) software. (D) Wild-type and Bcl-
2–expressing cells were grown in complete
media or media lacking EGF for 24 h, lysed,
and subject to immunoblotting with ?-LC3
EGF Withdrawal induces autoph-
EGFR. (A) Control (LPCX) and EGFR-expressing cells were grown
attached (A) or suspended (S) for 24 h, lysed, and subject to immu-
noblotting with ?-LC3 and ?-tubulin. (B) Punctate GFP-LC3 in
detached MCF-10A cells stably expressing GFP-LC3 or GFP-LC3 ?
EGFR suspended for 24 h. Bar, 20 ?m. (C) Control (LPCX) and
EGFR-overexpressing cells grown attached (A) or suspended for the
indicated times, were lysed and subject to immunoblotting with
?-P-ERK1/2, ?-ERK1 ? 2 (D) Control (LPCX) and EGFR-overex-
pressing cells grown attached (A) or suspended for the indicated
times, lysed, and subjected to immunoblotting with ?-P-AMPK, and
?-AMPK?. (E) Control (LPCX) and EGFR-overexpressing cells
grown attached (A) or suspended for the indicated times, lysed, and
subjected to immunoblotting with ?-P-eIF2? and ?-eIF2?. (F) Con-
trol (pBABE) and Bcl-2–expressing cells were grown attached of
suspended for the indicated times, lysed, and subjected to immu-
noblotting for the indicated antibodies. In A, C, and D, the E/P ?
lanes indicate E64d and pepstatin A added directly to the culture 4 h
Detachment-induced autophagy in cells overexpressing
C. Fung et al.
Molecular Biology of the Cell 802
tophagy in mammalian cells (Meley et al., 2006; Hoyer-
Hansen et al., 2007; Liang et al., 2007). In addition, we found
the increased phosphorylation of eukaryotic initiation factor
2? on serine 51 (P-eIF2?), a stress-regulated translational
nutrient starvation and endoplasmic reticulum (ER) stress
(Talloczy et al., 2002; Kouroku et al., 2007). Similar to AMPK
activation, the phosphorylation of eIF2? was not reduced in
detached EGFR-expressing cells (Figure 6E). Remarkably, we
also found increased AMPK activation and phosphorylated
eIF2? in Bcl-2–expressing cells during ECM detachment (Fig-
ure 6F). Hence, even though EGFR and Bcl-2 protect detached
cells from apoptosis, multiple stress pathways continue to be
potently activated in cells overexpressing these prosurvival
Increased Luminal Cell Death in 3D Acini on Chemical
Because anoikis is a principal contributor to the selective
death of central cells during lumen formation, we hypothe-
sized that the autophagic vacuoles observed in the lumen
during morphogenesis were mitigating the stresses of ECM
detachment. To initially test this hypothesis, we measured
the rates of luminal cell death in acini following acute phar-
macological inhibition of autophagy. We treated acini with
3-methyladenine (3-MA), an established pharmacological in-
hibitor of early autophagosome formation. Acini derived
from wild-type and Bcl-2–expressing MCF-10A cells were
3D cultured for 6 d; at this time point, minimal luminal cell
death is observed (Debnath et al., 2002). On day 6, cultures
were treated with 3-MA, and cell death was measured on
subsequent days by staining with EtBr, a DNA-intercalat-
ing dye only incorporated into dying cells. We observed
increased EtBr staining within the centers of acini treated
with 3-MA (Figure 7A), which correlated with increased
cleaved-caspase-3–positive cells in the lumens of individ-
ual wild-type structures (Figure 7A). When we enumer-
ated the percentage of EtBr-positive acini in both control
and 3-MA–treated cultures, we found increased luminal
cell death in cultures treated with this autophagy inhibi-
tor (Figure 7B). Similar results were obtained when day 6
acini were acutely treated with the lysosomal inhibitors
chloroquine (CQ) or bafilomycin A (BafA) (Figure 7C).
Finally, although Bcl-2 potently suppresses luminal cell
death, increased numbers of EtBr-positive acini were ob-
served in Bcl-2–expressing cultures treated with both au-
tophagy and lysosomal inhibitors (Figure 7, A–C).
Effect of ATG5 or ATG7 Depletion on Luminal Apoptosis
and Lumen Formation
In addition, we interrogated how the stable depletion of
ATG5 or ATG7 affected apoptosis and luminal filling in 3D
culture. MCF-10A cells expressing two independent small
hairpin RNAs against ATG5 (shATG5 hp 1 and 2) or ATG7
(shATG7 hp1 and 2) were generated. In cells expressing
these hairpins, we confirmed the stable reduction of the
ATG5:12 complex or ATG7 (Figure 8A). In these cells, we
observed potent knockdown for ?30 d, rendering them
suitable for use in long-term 3D morphogenesis assays.
Acini derived from shATG5- and shATG7-expressing cells
formed polarized structures that morphologically resembled
observed high levels of cleaved caspase-3 within these struc-
tures; in fact, the lumens of both ATG5 and ATG7 depleted
acini contained increased cleaved caspase-3–positive cells com-
pared with controls (Figure 8, C and D). Finally, to examine
whether the combined reduction of autophagy and apoptosis
would elicit the long-term survival of matrix-detached cells
occupying the lumens of 3D acini, we generated cells with
Bcl-2 overexpression plus stable ATG5 or ATG7 knockdown.
However, we did not observe significant luminal filling in cells
coexpressing Bcl2 ? shATG5 (Figure 9A) or Bcl2 ? shATG7
(Figure 9B) over 20 d of 3D culture. Similar to both wild-type
and Bcl-2–expressing controls, these structures exhibited a
hollow lumen. Thus, ATG5 or ATG7 depletion does not
cooperate with Bcl-2 to fill the lumen.
Here, we demonstrate that ECM detachment induces auto-
phagy in various nontumorigenic epithelial cell lines and in
autophagy inhibition. (A) Indicated cell types were cultured for 6 d
on Matrigel upon which 3-MA (10 mM) was added for 2 d. Repre-
sentative fields of EtBr stained day 8 acini (middle) and correspond-
ing phase contrast images (left) are shown. Bar, 20 ?m. Right, day 8
acini were immunostained with ?-cleaved caspase-3 (green) and
?-laminin-5 (red). DAPI-stained equatorial confocal cross-sections
are shown. Bar, 20 ?m. (B) After 6 d of 3D culture, 3-MA (10 mM)
was added to the indicated cell types; the percentage of acini con-
taining EtBr-positive cells was enumerated on subsequent days.
Results are the mean ? SEM of four independent experiments. (C)
After 6 d of 3D culture, CQ (20 ?M) or BafA (20 nM) was added to
the indicated cell types; the percentage of acini containing EtBr-
positive cells was enumerated on subsequent days. Results are the
mean ? SEM of three independent experiments.
Increased luminal cell death in 3D acini upon chemical
Autophagy during Anoikis
Vol. 19, March 2008803
primary human epithelial cells. We also discover that ATG
depletion results in both enhanced cleaved caspase-3 during
anoikis and reduced clonogenic viability upon reattachment.
Remarkably, matrix-detached cells still exhibit autophagy
when apoptosis is blocked by Bcl-2 expression, and ATG
depletion impairs the clonogenic survival of detached Bcl-
2–expressing cells. Thus, autophagy promotes epithelial cell
survival during anoikis, including detached cells harboring
Anoikis serves as an important mechanism to maintain
tissue homeostasis by killing cells that have lost contact with
an underlying basement membrane (Gilmore, 2005). How-
ever, evidence indicates that the detachment of nonmalig-
nant cells also triggers antiantiapoptotic signals, such as
nuclear factor-?B and inhibitor of apoptosis protein family
members; these antiapoptotic mechanisms presumably de-
lay the onset of apoptosis and allow cells to survive if they
are able to reestablish ECM contact in a timely manner (Yan
et al., 2005; Liu et al., 2006). Our results indicate, similar to
these antiapoptotic pathways, the induction of autophagy
also contributes to cell viability during ECM detachment.
Overall, our results resemble other circumstances where
autophagy protects cells exposed to various forms of duress,
including nutrient deprivation, growth factor withdrawal,
ER stress, and ischemia (Boya et al., 2005; Lum et al., 2005;
Degenhardt et al., 2006; Ogata et al., 2006; Kouroku et al.,
2007). Autophagy may promote cell survival by a number of
mechanisms, such as 1) generating nutrients and energy to
sustain starving or stressed cells, 2) degrading toxic proteins,
or 3) protecting cells from oxidative stress by sequestering
damaged mitochondria (Debnath et al., 2005; Levine and
Yuan, 2005). Further studies are required to identify the
precise mechanisms through which detachment-induced au-
tophagy promotes cell survival. Moreover, in addition to
regulating survival, autophagy may direct other critical bi-
ological functions during matrix detachment; for example,
autophagy suppresses both DNA damage and chromosomal
depletion on luminal apoptosis. (A) Top,
MCF-10A cells were infected with retrovirus
encoding an empty control vector (pSR) or
two independent small hairpin RNAs to
knockdown ATG5 (shATG5 hp1 and 2).
Bottom, MCF-10A cells were infected with
lentiviral particles expressing a nontarget-
ing hairpin (shCntrl) or two independent
small hairpin RNAs to knockdown ATG7
(shATG7 hp1 and 2). Lysates were immuno-
blotted with ?-ATG7 and ?-tubulin. (B) Rep-
resentative phase contrast images of acini
generated from control (pSR or shCntrl),
shATG5 (hp 1 and 2), and shATG7 (hp1 and
2) cells after 18 d of 3D culture. Bar, 20 ?m.
(C) Indicated cell types grown in 3D for
12 d were immunostained for ?-cleaved
caspase-3 (green), ?-laminin-5 (red), and
counterstained with DAPI to detect nuclei
(blue). Confocal cross sections of equators
are shown. Bar, 20 ?M. (D) Acini from the
indicated cell types were fixed and immu-
nostained on day 12 as illustrated in C. Cells
occupying the lumen were defined as those
lacking direct contact with basement mem-
brane as delineated by laminin 5 staining.
The percentage of cells positive for cleaved
Effect of Stable ATG5 or ATG7
caspase-3 (mean ? SEM) was counted in 99 acini obtained from three independent 3D culture experiments.
formation. (A and B) Day 20 acini from the indicated cell types
grown were immunostained for ?-cleaved caspase-3 (green),
?-laminin-5 (red), and counterstained with DAPI to detect nuclei
(blue). Confocal equatorial cross-sections are shown. Bar, 20 ?m.
Effect of stable ATG5 or ATG7 depletion on lumen
C. Fung et al.
Molecular Biology of the Cell 804
instability in response to metabolic stress (Karantza-Wads-
worth et al., 2007; Mathew et al., 2007).
Because integrin engagement is critical for the proper
transduction of growth factor receptor mediated signals, we
hypothesized that detachment-induced autophagy results
from reduced growth factor receptor signaling. Importantly,
in human mammary epithelial cells, ECM contact is required
for both EGFR expression and the activation of critical
downstream signals (Reginato et al., 2003). However, we
demonstrate that even though enforced EGFR expression
during anoikis can restore certain downstream survival-
promoting signals, such as ERK activation, it is not sufficient
to prevent detachment-induced autophagy. Thus, we postu-
late that other cell adhesion regulated stress pathways also
direct detachment-induced autophagy. We have identified
multiple signals during ECM detachment that may posi-
tively regulate autophagy, including AMPK activation and
the phosphorylation of eIF2?; in ongoing studies, we are
trying to dissect how these signals, either individually or in
combination, contribute to detachment-induced autophagy.
Remarkably, a recent study demonstrates that integrin
engagement, most notably ?3?1, is actually required to sus-
tain autophagy in starved prostate epithelial cells (Edick et
al., 2007). In this study, the observed reductions in autoph-
agy were based exclusively on measurements of GFP-LC3
puncta; thus, it remains unknown whether the reductions in
punctate GFP-LC3 observed upon integrin blockade are ac-
tually due to decreased autophagosome formation versus
increased LC3-II turnover in the lysosome (Tanida et al.,
2005). Alternatively, detachment-induced autophagy may
be cell type or context dependent. Importantly, whereas
human mammary epithelial cells die after 24–48 h of de-
tachment, certain epithelial cells, notably primary mouse
mammary epithelial cells and rat intestinal epithelial cells,
perish within a few hours following substratum detach-
ment; thus, one can speculate that detachment-induced au-
tophagy will not contribute to the viability of cells that
undergo rapid anoikis (Gilmore, 2005).
Protection from anoikis is thought to promote filling of the
normally hollow lumen in glandular epithelial structures, a
hallmark of early epithelial cancers, such as carcinomas in
situ (Debnath and Brugge, 2005; Gilmore, 2005). Although
our previous work suggested that cells undergo autophagy
as they die during 3D morphogenesis, two results in this
study argue against a direct role for type 2 death during
lumen formation. First, the acute pharmacological inhibition
of autophagy during morphogenesis enhances luminal cell
death. Second, the knockdown of ATG5 or ATG7, two pro-
teins critical for early autophagosome formation, enhances
luminal apoptosis during MCF-10A 3D morphogenesis and
fails to elicit long-term luminal filling, even when combined
with apoptosis inhibition. Our results are consistent with
recent studies in E1A immortalized mouse mammary cells
lacking one allele of Beclin; when grown in 3D culture, cells
with reduced Beclin exhibit increased lumen formation com-
pared with wild-type controls (Karantza-Wadsworth et al.,
2007). Interestingly, in embryoid bodies derived from cells
lacking atg5 or beclin1, apoptotic cell corpses fail to clear
during cavitation, because autophagy is critical for the pre-
sentation of signals, such as phosphatidylserine, that medi-
ate the phagocytic clearance of apoptotic cells (Qu et al.,
2007). Accordingly, the increased numbers of apoptotic cells
that we observe when autophagy is inhibited during lumen
formation could arise from defective corpse clearance. How-
ever, over longer times, we do not observe reduced clear-
ance in ATG5 or ATG7-depleted structures; rather, these
structures form hollow lumens at rates similar to controls.
Clearance may ultimately proceed during 3D morphogene-
sis because we have only reduced these ATGs rather than
completely eliminated these proteins in acini. Nonetheless,
recent work indicates that inhibition of autophagy, a late
stage event observed in MCF7 carcinoma cells during
anoikis, does not prevent phagocytic engulfment (Petrovski
et al., 2007). Importantly, during both 3D lumen formation
and ECM detachment, we have observed the robust induc-
tion of autophagy in Bcl-2–expressing cells, indicating that
autophagy can proceed independently of apoptosis in ECM-
detached cells, rather than merely serve as a secondary
clearance mechanism to remove cells undergoing apoptosis
(Debnath et al., 2002). Further delineating the respective
roles of autophagy in luminal cell survival versus the phago-
cytic clearance of dying cells during lumen formation re-
mains a topic for future investigation.
Several autophagy regulators are down-regulated in hu-
man cancers, including BECN-1/ATG6, which is monoalleli-
cally deleted in 40–75% of breast, prostate, and ovarian
tumors, and Death Associated Protein Kinase 1, which is meth-
ylated in many tumors (Liang et al., 1999; Inbal et al., 2002).
The importance of autophagy as a tumor suppressor is
further supported by the development of tumors in becn-1?/?
mice (Qu et al., 2003; Yue et al., 2003). In contrast, because
autophagy has well-established cytoprotective functions, it
can promote the survival of tumor cells exposed to stresses
such as hypoxia, nutrient limitation, and chemotherapy
(Ogier-Denis and Codogno, 2003; Jin et al., 2007). Both of
these opposing functions may be relevant to cancer progres-
sion and treatment. Our results broach the possibility that
autophagy contributes to the survival of oncogenic cells
lacking appropriate matrix contact. Indeed, the ability to
survive in the absence of normal ECM is considered a critical
feature of metastasis, because cancer cells in the bloodstream
or secondary tissue sites are either deprived of matrix or
exposed to foreign matrix components (Chambers et al.,
2002). Accordingly, we are currently investigating how on-
cogenic pathways regulate autophagy during ECM detach-
ment and determining whether autophagy contributes to the
survival and expansion of oncogene-expressing cells under-
going anoikis and in the lumens of 3D structures.
We are especially grateful to Dr. Joan Brugge (Harvard Medical School), in
whose laboratory this work was initiated. Drs. Noburu Mizushima and
Tamotsu Yoshimori generously provided reagents and cells. Grant support
includes National Institutes of Health KO8 Award (CA-098419), Culpeper
Scholar Award (Partnership for Cures), an American Association for Cancer
Research/Genentech BioOncology Career Award, a Howard Hughes Medical
Institute Early Career Award, and funds from the UCSF Sandler Program in
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