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-
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Molecular Biology of the Cell 806