The Journal of Cell Biology
The Journal of Cell Biology, Volume 163, Number 6, December 22, 2003 1397–1407
The Rockefeller University Press, 0021-9525/2003/12/1397/11 $8.00
Autocrine laminin-5 ligates
RAC and NF
B to mediate anchorage-independent
survival of mammary tumors
4 integrin and activates
Gabriela I. Rozenberg,
Johnathon N. Lakins,
M. Peter Marinkovich,
and Valerie M. Weaver
Department of Pathology and Laboratory Medicine, School of Medicine and
Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104
Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305
Department of Cell Biology, Cytokinetics, Inc., South San Francisco, CA 94080
Wells Center for Pediatrics Research, Indiana University School of Medicine, Indianapolis, IN 46202
Department of Bioengineering, Institute for
nvasive carcinomas survive and evade apoptosis despite
the absence of an exogenous basement membrane. How
epithelial tumors acquire anchorage independence for
survival remains poorly defined. Epithelial tumors often
secrete abundant amounts of the extracellular matrix protein
laminin 5 (LM-5) and frequently express
Here, we show that autocrine LM-5 mediates anchorage-
independent survival in breast tumors through ligation of a
wild-type, but not a cytoplasmic tail–truncated
4 integrin does not mediate tumor survival through
activation of ERK or AKT. Instead, the cytoplasmic tail of
integrin is necessary for basal and epidermal growth factor–
induced RAC activity, and RAC mediates tumor survival.
Indeed, a constitutively active RAC sustains the viability of
mammary tumors lacking functional
through activation of NF
B, and overexpression of NF
p65 mediates anchorage-independent survival of nonmalig-
nant mammary epithelial cells. Therefore, epithelial tumors
could survive in the absence of exogenous basement
membrane through autocrine LM-5–
Malignant transformation is linked to the migration of
transformed epithelial cells across the endogenous basement
membrane (BM) and their survival and proliferation in the
surrounding interstitial collagen-rich stroma. Because normal
mammary epithelial cells (MECs) require ligation of BM
receptors to grow and survive (Weaver et al., 1997), invasive
breast cancers must be able to resist apoptosis. Consistently,
endogenous apoptosis rates decrease as MECs transition
from ductal carcinoma in situ to invasive carcinoma
(Gandhi et al., 1998), and immortalized mammary tumor
cells frequently exhibit apoptosis resistance (Fernandez et al.,
2002). However, how transformed MECs acquire apoptosis
resistance remains poorly understood.
Breast tumors and immortalized mammary tumor cells
frequently lose expression of
which are the BM receptors that support normal MEC
growth, differentiation, and survival (Weaver et al., 1996).
Mammary tumors also express high amounts of antiapoptotic
proteins such as activated focal adhesion kinase (Cance et al.,
B (Sovak et al., 1997), bcl-2 (Kalogeraki et al.,
2002), and survivin (Tanaka et al., 2000), and often have
elevated levels of active prosurvival kinases such as ERK, PI 3,
and AKT (Fukazawa et al., 2002). Therefore, apoptosis-
resistant breast tumors could arise through selection of
MECs that express sufficient quantities of antiapoptotic
molecules that permit growth and survival in the absence of
adhesion (anoikis; Frisch and Ruoslahti, 1997).
Primary human breast tumors frequently synthesize and
secrete ECM proteins such as fibronectin (Ioachim et al.,
Address correspondence to Valerie M. Weaver, Institute for Medicine
and Engineering, University of Pennsylvania, 1170 Vagelos Research
Laboratory, 3340 Smith Walk, Philadelphia, PA 19104-6383. Tel.:
(215) 573-7389. Fax: (215) 573-6815.
Key words: mammary epithelial cell;
4 integrin; apoptosis; GTPase;
Abbreviations used in this paper: 2D, two-dimensional; 3D, three-
BM, basement membrane; LM-5, laminin 5; MEC, mammary epithelial
cell; rBM, reconstituted BM.
1398 The Journal of Cell Biology
Volume 163, Number 6, 2003
2002) and laminin-5 (LM-5; Davis et al., 2001). Malignant
MECs often up-regulate
al., 1993; Pena et al., 1994) and retain
pression (Davis et al., 2001). Ligation of
fibronectin (Nista et al., 1997) and
(Bachelder et al., 1999) supports cell growth and survi-
val. Thus, apoptosis-resistant mammary tumors could arise
through increased growth and survival of MECs that possess
enhanced autocrine ECM–integrin signaling, although such
a possibility has yet to be investigated.
We have been studying the role of MEC–ECM interac-
tions and apoptosis resistance in the pathogenesis of breast
cancer using the HMT-3522 human tumor progression
model (Weaver et al., 1996). Analogous to breast tumor
progression in vivo, the early passage, nonmalignant cells in
this series (S-1) require ligation of
for their growth and survival, whereas their tumorigenic
progeny (T4-2) are anchorage independent (Wang et al.,
1998). Instead of dying, in the absence of
tion, the T4-2s revert to form polarized tissue structures that
resemble nonmalignant acini (Weaver et al., 1997). We
4 integrin can mediate apoptosis re-
sistance to exogenous apoptotic stimuli in three-dimensional
(3D) tissues irrespective of growth and malignancy status if
MEC tissues are polarized (Weaver et al., 2002). Because in-
vasive breast tumors typically lose polarized tissue architec-
ture (acini, ductal) and tumors often metastasize as isolated
cells, we asked whether
4 integrin might also support
the survival of mammary tumor cells lacking polar tissue
v integrins (Koukoulis et
4 integrin ex-
1 integrin by
4 integrin by LM-5
1 integrin liga-
structure, and if so, how. Here, we report that nonpolarized
malignant MECs grown as 3D structures survive via
integrin, provided they synthesize and secrete sufficient
quantities of LM-5 and activate RAC and NF
4 integrin mediates anchorage-independent
survival of mammary tumors
4 integrin drives tumor invasion and migration (Mercu-
rio and Rabinovitz, 2001), and mediates apoptosis resistance
in polarized MEC acini (Weaver et al., 2002), suggesting
that tumors that express
and acquire multi-drug resistance, provided they are able to
recapitulate tissue polarity. Because tumor invasion requires
loss of tissue integrity, and survival of individual and isolated
clusters of tumor cells dictates metastatic efficiency (Wong
et al., 2001), we investigated whether
also support the growth and survival of isolated tumor cells
or disorganized tumor cell clusters. We used S-1 (nonmalig-
nant) and T4-2 (tumorigenic) MECs from the HMT-3522
human mammary tumor progression model and investi-
gated whether anchorage independence of the T4-2s de-
We found that total and cell surface
expression are higher in the T4-2s compared with S-1s, but
2 integrin levels remain constant (Fig. 1 A and Fig. 3
A; Weaver et al., 1997). We also determined that although
isolated S-1s require
1 integrin ligation for growth and sur-
4 integrin could metastasize
4 integrin could
showing increased ?4 integrin in the T4-2s compared with S-1s (S-1, 265 vs. T4-2, 430) and similar amounts of ?2 integrin. (B) Apoptotic
labeling indices were calculated using the TUNEL assay in S-1s and T4-2s grown in rBM for 96 h in the presence of function-blocking mAb
against ?1 integrin (AIIB2) and/or ?4 integrin (ASC-3). Results are the mean ? SEM of at least three separate experiments. (C) Phase-contrast
(c), conventional immunofluorescence (EGFP; c?) and confocal Immunofluorescence microscopy of ?6 integrin (Texas red; c??), ?4 integrin
(EGFP; c???), and ?6 and ?4 integrin overlay (yellow; cIV, as indicated by white and black arrows) showing uniform inducible expression of
tailless EGFP ?4 integrin (T4 ?4?cyto) in T4-2s and clustering of ?6/?4?cyto integrin at membrane adhesion plaques. Bars: (c and c?) 50 ?m;
(c??–cIV) 20 ?m. (D) Relative cell adhesion levels calculated using a fluorescence assay of control T4-2s (T4-2), and T4 ?4?cyto cells showing
comparable adhesion to rBM and LM-5 in both cell types. (E) FACS® analysis showing similar levels of the LM integrins ?1, ?4, ?3, and ?6 in
T4-2 and T4 ?4?cyto cells. (F) Cell viability was calculated using the Live/Dead assay for T4-2 or T4 ?4?cyto cells grown in rBM for 96 h
with or without a function-blocking mAb to ?1 integrin. (G) Soft agar assay results demonstrating that expression of the tailless ?4 integrin
(T4 ?4?cyto) inhibits anchorage-independent growth of T4-2s, so that infected cells behave like S-1 nonmalignant cells (S-1). Results for B,
D, F, and G are the mean ? SEM of three to four separate experiments. (B and F) *, P ? 0.05. (G) **, P ? 0.01.
?6?4 integrin mediates anchorage-independent survival of mammary tumors. (A) FACS® analysis of cell surface integrin levels
Breast tumors survive via ?4 integrin, RAC, and NF?B | Zahir et al. 1399
vival, and T4-2s do not (Fig. 1 B), in the absence of both ?1
and ?4 integrin ligation, T4-2s die (Fig. 1 B). Thus, growth
and survival of T4-2s require activation of either ?4 or ?1
Although much is known about how ?1 integrin het-
erodimers mediate cell viability, much less is known about
how ?6?4 integrin directs cell survival. The cytoplasmic
tail of ?4 integrin mediates proliferation through ras
(Dans et al., 2001), invasion and survival via PI 3-kinase
(Mercurio and Rabinovitz, 2001), and cell polarity via
hemidesmosome formation (Weaver et al., 2002). To ex-
plore how ?6?4 integrin regulates MEC survival, we se-
lected pooled populations of T4-2s that stably expressed
high levels of a doxycyclin-repressible, EGFP-tagged, tail-
less ?4 integrin (?4?cyto; Fig. 1 C, c?), which colocalizes
with ?6 integrin at adhesion plaques (Fig. 1 C, c??, c???,
and cIV), equally supports adhesion to BM and purified
LM-5 when compared with wild-type ?4 integrin (T4-2
expressing ?4WT; Fig. 1 D), and has no effect on plasma
membrane levels of the LM integrins ?1, ?4, ?3, and ?6
(Fig. 1 E). We found that T4-2s expressing the EGFP-
tagged ?4?cyto required ?1 integrin ligation for their sur-
vival (Fig. 1 F) and failed to form colonies in soft agar
(Fig. 1 G). This indicates that ?6?4 integrin cytoplasmic
function is required for the anchorage-independent sur-
vival phenotype of these tumors.
Increased ?4 integrin does not induce
anchorage-independent survival of
Because altering ?4 integrin activity had such a profound ef-
fect on tumor growth and survival, we asked whether over-
expression of a ?4WT would be sufficient to confer anchor-
age independence to nonmalignant MECs (S-1 cells). S-1
cells were infected with ?4WT, and selected pooled popula-
tions of MECs expressing elevated levels of total (Fig. 2 A)
and membrane-localized ?6?4 integrin (Fig. 2 B) were used
for experiments. Control S-1s grown in 3D reconstituted
BMs (rBMs) died rapidly when ?1 integrin–BM interac-
tions were inhibited (Fig. 2 C; Weaver et al., 1997). How-
ever, S-1s overexpressing ?4WT remained viable despite the
absence of ?1 integrin–BM interactions (Fig. 2 C), indicat-
ing that increased activity of ?6?4 integrin can sustain the
growth and survival of nonmalignant MECs. Yet, S-1s over-
expressing ?4WT died if ?1 integrin signaling was inhibited
when viability experiments were conducted in 3D collagen I
gels where exogenous LM (?6?4 integrin ligand) was absent
(Fig. 2 D). Moreover, S-1 ?4WT MECs failed to grow in
soft agar (Fig. 2 E, compare S-1 ?4WT with S-1 control
with T4-2). Therefore, a signal functionally linked to the cy-
toplasmic tail of ligated ?4 integrin supports the survival of
isolated and nonpolarized clusters of nonmalignant MECs
grown in 3D.
Autocrine LM-5 mediates ?4 integrin–dependent
survival of mammary tumors
Breast tumors synthesize and secrete abundant quantities of
LM-5 (Davis et al., 2001). Because we found that ?6?4 in-
tegrin supports anchorage independence of T4-2s but not of
S-1s, we postulated that malignant transformation is either
associated with constitutive activation of ?6?4 integrin or
is linked to increased synthesis and secretion of autocrine
LM-5. Consistent with the latter prediction, T4-2s have ele-
vated levels of total ?4 integrin (Fig. 3 A) and plasma mem-
brane ?4 integrin (Fig. 1 A), and synthesize and secrete
more LM-5 than S-1s in 3D ECM gels (Fig. 3 B). More-
over, T4-2s colonies in soft agar are surrounded by copious
amounts of LM-5 (Fig. 3 D), and when LM-5–?6?4 inte-
grin interactions and ?1 integrin ligation are simultaneously
blocked (using function-blocking mAbs) in tumors embed-
ded within a collagen I gel (in the absence of an exogenous
?6?4 integrin ligand), T4-2s die (Fig. 3 C). Consistently,
although S-1s and S-1 ?4WT cells embedded within 3D
collagen I gels die when ?1 integrin ligation is inhibited, S-1
?4WT, but not control S-1s, grow and survive significantly
better in the presence of exogenous purified LM-5 (Fig. 3
independent growth and survival of nonmalignant MECs. (A) Immu-
noblot analysis of total RIPA lysates for S-1 controls (S-1) and S-1 cells
overexpressing a full-length ?4 integrin (S-1 ?4WT) demonstrating
increased expression of total ?4 integrin in S-1 ?4WT cells. (B) FACS®
analysis of membrane ?1 integrin and ?4 integrin in S-1s and S-1
?4WT showing elevated ?4 integrin expression in the infectants
(S-1, 260 vs. S-1 ?4WT, 650) and no effect on ?1 integrin. (C) Cell
viability was calculated using the Live/Dead assay for S-1 and S-1
?4WT grown in rBM for 96 h with and without a function-blocking
mAb to ?1 integrin. (D) Percent apoptosis was calculated by scoring
the number of caspase 3–positive cells for S-1 and S-1 ?4WT grown
in collagen I for 96 h with and without function-blocking mAb to
?1 integrin. (E) Soft agar assay results demonstrating that whereas
malignant T4-2s exhibit anchorage independent growth and survival
S-1s do not, even if they overexpress ?4 integrin (S-1 ?4WT). Results
for C–E are the mean ? SEM of three to four separate experiments.
*, P ? 0.05.
?4 integrin overexpression does not induce anchorage-
1400 The Journal of Cell Biology | Volume 163, Number 6, 2003
D). Thus, tumors grow and survive in the absence of an ex-
ogenous BM if they synthesize and secrete sufficient quanti-
ties of LM-5 and up-regulate and ligate ?6?4 integrin.
?6?4 integrin does not require ERK or AKT to mediate
How does LM-5 ligation of ?6?4 integrin induce tumor
survival? ?6?4 integrin can mediate the survival of tumor
cells through activation of PI 3-kinase and AKT (Bachelder
et al., 1999). Ligation of ?6?4 integrin also activates ERK
via SHC-dependent activation of ras (Dans et al., 2001),
and ERK supports anchorage-independent survival in epi-
thelial cells (Howe et al., 2002). Although treatment with
PD98059 effectively repressed ERK activity (Fig. 4 B), T4-2
survival remained unaffected, even when ?1 integrin liga-
tion was simultaneously blocked (Fig. 4 A). Likewise, treat-
ment of T4-2s with LY294002 inhibited AKT activity (Fig.
4 B), yet failed to compromise ?1 integrin-independent
growth and survival (Fig. 4 A). Indeed, concomitant inhibi-
tion of ERK, PI 3-kinase, and ?1 integrin activity had no
appreciable effect on tumor viability (Fig. 4 A).
Because AKT is an oncogene that can mediate adhesion-
dependent survival in tumors (Hill and Hemmings, 2002),
we directly tested the importance of AKT activity to ?6?4
integrin–dependent survival by expressing a dominant-nega-
tive K179M AKT. Despite stable expression of high levels of
HA-tagged dominant-negative AKT (Fig. 4 D), T4-2s re-
mained completely viable (Fig. 4 C) irrespective of their ?1
integrin ligation status, even when ERK activity was also in-
hibited (Fig. 4 C). Conversely, stable expression of the same
dominant-negative AKT (Fig. 4 F) modestly but signifi-
cantly compromised the viability of isolated S-1s embedded
within rBM (Fig. 4 E). Consistently, stable overexpression
of high levels of a constitutively active HA-tagged myristoy-
lated AKT (Fig. 4 H, MyrAkt) only partially rescued S-1
survival when ?1 integrin ligation was blocked (Fig. 4 G),
whereas overexpression and ligation of ?6?4 integrin was
significantly more effective (Fig. 2 C). Thus, ?6?4 integrin
must be able to support MEC survival through pathways
that are distinct from AKT and ERK.
?4 integrin mediates tumor survival through
regulation of RAC
The Rho GTPase RAC is frequently overexpressed in tu-
mors of the breast (Fritz et al., 1999), permits MEC growth
in soft agar (Bouzahzah et al., 2001), and protects MDCK
cells from anoikis (Coniglio et al., 2001). Because neither in-
hibition of AKT nor ERK kinase compromised T4-2 sur-
vival (Fig. 4, A–D), we asked whether ?6?4 integrin medi-
ated T4-2 survival via activation of the Rho GTPase RAC.
We assayed for RAC activity and determined that both basal
and EGF-induced RAC activity significantly correlated with
levels of expressed and ligated ?6?4 integrin. For example,
T4-2s that express high ?6?4 integrin also have increased
basal and EGF-induced RAC activity relative to S-1s (Fig. 5,
A and B, compare specific activity of RAC in T4-2 with S-1;
and Fig. 5, C and D, EGF-stimulated RAC activity). More-
over, ablating ?6?4 integrin function in the T4-2s by ex-
pressing the dominant-negative ?4?cyto, reduced both
basal (Fig. 5, A and B) and EGF-stimulated (Fig. 5 D) RAC
activity, and overexpression of ?4WT in the S-1 cells led to
an increase in both basal (Fig. 5, A and B) and EGF-stimu-
lated RAC activity (Fig. 5 C).
To determine whether ?6?4 integrin mediated anchor-
age-independent survival in T4-2s through RAC, we exam-
ined the survival phenotype of S-1 and T4-2s that expressed
constitutively active and dominant-negative RhoGTPase
mutants. Pooled populations of T4-2s stably expressing
dominant-negative EGFP-N17 RAC (Fig. 6 A) had reduced
RAC activity (unpublished data) and required ?1 integrin
ligation for survival (Fig. 6 B), despite high levels of BM-
ligated ?6?4 integrin (Figs. 1–3). S-1s that expressed low
levels of endogenous ?4 integrin (Figs. 1–3) no longer de-
pended on ?1 integrin activity for survival and were able to
of mammary tumors. (A) Immunoblot analysis of total cell lysate and
immunoprecipitants of secreted protein showing increased cellular
?4 integrin and secreted LM-5 in the T4-2s compared with S-1s.
Note that E-cadherin levels remain constant regardless of the state of
cell transformation. (B) Confocal Immunofluorescence microscopy
images of ?4 integrin, LM-5, and collagen IV (Coll IV) in S-1 and T4-2
3D tissue structures. Data indicate that after malignant transformation,
tumors have increased expression of cell surface ?4 integrin and
secrete more extracellular LM-5, whereas collagen IV deposition
does not change appreciably. All cultures were analyzed after 10 d
inside the rBM. Bar, 20 ?m. (C) Apoptotic labeling indices calculated
using the TUNEL assay in T4-2s grown in collagen I for 96 h in the
presence or absence of function-blocking mAb against ?1 integrin
(AIIB2) and/or LM-5 (BM165). (D) Phase-contrast microscopy images
of a representative T4-2 colony in soft agar (d) showing significant
amounts of LM-5 deposition (d?; HRP) and specificity of staining in a
parallel colony (d??) treated without primary mAb (d???). Bar, 50 ?m.
(E) Percent apoptosis was calculated by scoring the number of
caspase 3–positive S-1 and S-1 ?4WT cells grown in collagen I for
96 h with or without 10 ?g/ml of exogenous LM-5 and/or function-
blocking mAb to ?1 integrin. Results in C and E are the mean ? SEM
of at least three separate experiments. *, P ? 0.05
Autocrine LM-5 mediates anchorage-independent survival
Breast tumors survive via ?4 integrin, RAC, and NF?B | Zahir et al. 1401
grow in soft agar (Fig. 6, F and G) if they stably expressed
the constitutively active c-myc-V12 RAC (Fig. 6 E). Because
T4-2s stably expressing a dominant-negative EGFP N19
Rho retained their apoptosis-resistant phenotype (Fig. 6, C
and D), we suggest that LM-5–ligated ?6?4 integrin medi-
ates mammary survival through RAC.
?6?4 integrin mediates tumor survival via
RAC-dependent activation of NF?B
Having established a link between ?6?4 integrin, RAC,
and survival, we next sought to delineate the mechanism
whereby RAC mediates MEC survival. RAC can activate
NF?B p65 (Bouzahzah et al., 2001), and we showed that
?6?4 integrin induces apoptosis resistance in acini through
NF?B (Weaver et al., 2002). Upon investigation, we found
that S-1s expressing c-myc-V12RAC had high amounts of
nuclear NF?B (Fig. 7, A and B) and that treating reverted
T4-2 acini, which exhibit constitutively active NF?B (Fig.
survival in mammary tumors via ERK or AKT. (A) Percent apoptosis
calculated by scoring the number of caspase 3–positive T4-2s grown in
rBM for 96 h with or without a function-blocking mAb to ?1 integrin
and treatment with 50 ?M of the PI 3-kinase inhibitor LY 294002
(LY), 20 ?M of the MEK inhibitor PD 98059 (PD), or vehicle (DMSO;
Vehicle). (B) Immunoblot analysis of total and activated ERK and
AKT in T4-2s grown in rBM with or without LY 294002 and/or PD
98059 treatment, was as described in A. (C) Percent apoptosis was
calculated as described in A for T4-2 Vector control (Vector) and
T4-2s expressing a dominant-negative AKT (DNAkt) grown in rBM
for 96 h with or without a function-blocking mAb to ?1 integrin and
treatment with the MEK inhibitor PD 98059 (PD) as indicated. (D)
Immunoblot analysis of HA expressed in control T4-2s (Vector) and
T4-2s expressing a HA-tagged dominant-negative AKT (DNAkt). (E)
Percent apoptosis calculated as in A for S-1 control (Vector) and S-1s
expressing a dominant-negative AKT (DNAkt) grown in rBM for 96 h.
Note that expression of the dominant-negative AKT decreased survival
of S-1s yet failed to compromise the viability of the T4-2s, even when
?1 integrin and ERK activity were inhibited. (F) Immunoblot analysis
of HA in RIPA lysates of S-1 controls (Vector) and S-1s expressing an
HA-tagged dominant-negative AKT (DNAkt). (G) Percent apoptosis
was calculated as in A for S-1 vector control (Vector) and S-1s
expressing a constitutively active myristoylated AKT (S1 MyrAkt)
grown in rBM for 96 h with or without a function-blocking mAb to
?6?4 integrin does not mediate anchorage-independent
?1 integrin. Data indicate that although active AKT does significantly
enhance anchorage-independent survival of nonmalignant MECs,
it does not completely rescue S-1 viability. (H) Immunoblot analysis
of HA in S-1 controls (Vector) or S-1s expressing an HA-tagged
constitutively active myristoylated AKT (MyrAkt). All apoptosis
data are the mean ? SEM of at least three separate experiments.
**, P ? 0.01; ***, P ? 0.001.
sentative immunoblot of immunoprecipitated PAK-associated RAC
(GTP-Rac), total cellular RAC (Rac) and total E-cadherin in S-1
control, T4-2 control, S-1s overexpressing ?4 integrin (S-1 ?4WT),
and T4-2s expressing a tailless ?4 integrin (T4 ?4?cyto). (B) Relative
specific activity of RAC in S-1s, T4-2s, S-1 ?4WT, and T4 ?4?cyto
cells was calculated by densitometric analysis of immunoblots of
activated (PAK-associated) RAC divided by total cellular RAC after
normalization to total E-cadherin. Results are the mean ? SEM of
three to five separate experiments. *, P ? 0.05; **, P ? 0.001. (C) Time
course of EGF-induced RAC activation , detected as described in A,
in S-1s and S-1 ?4WT cells. Data show significantly enhanced EGF-
induced RAC activation in S-1s expressing higher levels of ligated
?4 integrin. (D) Time course of EGF-induced RAC activation, detected
as described in A, in T4-2s and T4 ?4?cyto cells showing a significant
reduction in EGF-induced RAC activation in T4-2s lacking the
cytoplasmic tail of ?4 integrin. Time course results show one repre-
sentative experiment out of four.
?6?4 integrin regulates RAC activity in MECs. (A) Repre-
1402 The Journal of Cell Biology | Volume 163, Number 6, 2003
7, C and D; Weaver et al., 2002), with the Rho GTPase in-
hibitor Toxin A difficile repressed nuclear NF?B signifi-
cantly (Fig. 7, C and D, compare T4?1 with T4?1 Toxin
A), inhibited RAC activity noticeably, disrupted actin orga-
nization appreciably, and eventually killed the T4-2 rever-
tants (unpublished data). Because expressing a dominant-
negative N19 Rho did not compromise the viability of T4-2
revertants (Fig. 6 D), whereas N17 RAC did (Fig. 6 B), we
conclude that the Toxin A phenotype was likely due to inhi-
bition of RAC.
Full-length ?4 integrin but not a ?4?cyto permits nu-
clear translocation (Fig. 7, E and F) and activation (Fig. 7
G) of NF?B in T4-2s. Therefore, we predicted that if ?6?4
integrin regulates NF?B activity via RAC, a constitutively
active RAC (V12 RAC) should confer anchorage-indepen-
dent survival to T4-2s expressing the ?4?cyto, and tumor
viability should depend on NF?B activation. Consistently,
T4-2s expressing both the ?4?cyto and a constitutively ac-
tive RAC (?4?cyto/V12 RAC) survived when ?1 integrin–
ECM interactions were blocked (Fig. 7 H), and tumor cells
expressing both transgenes regained their ability to form col-
onies in soft agar (Fig. 7 I). Furthermore, anchorage-inde-
pendent survival of the ?4?cyto/V12RAC-expressing T4-2s
absolutely required NF?B activity (Fig. 8 B). Indeed, T4-2s
expressing only the ?4?cyto died when ?1 integrin func-
tion was blocked (Fig. 7 H and Fig. 1 F) and T4-2 ?4?cyto
MECs failed to form colonies in soft agar (Fig. 7 I). There-
fore, anchorage-independent growth and survival of T4-2s
depends on a signaling pathway initiated through LM-5 li-
gation of ?6?4 integrin that is transduced by RAC and that
depends on NF?B activation.
NF?B activity is necessary and sufficient for
anchorage-independent survival of MECs
NF?B is induced in the early stages of mammary involu-
tion and its activation is associated with enhanced MEC
survival in culture (Clarkson et al., 2000). NF?B expres-
sion and activity are increased in mammary tumors (Sovak
et al., 1997). We have shown that NF?B mediates resis-
tance to chemotherapy, radiation treatment, and receptor-
induced apoptosis (Baldwin, 2001; Weaver et al., 2002).
To directly determine if ?6?4 integrin-dependent activa-
tion of NF?B is essential for the survival of T4-2 re-
vertants, we inhibited NF?B nuclear translocation and
assayed for effects on ?1 integrin–dependent survival. In-
cubation with the membrane-soluble peptide SN50 that
specifically inhibits nuclear translocation of NF?B, but not
the nonfunctional peptide SN50M, induced apoptosis in
the control T4-2 and T4 ?4?cyto/V12RAC revertants,
but had no effect on viability when ?1 integrin was ligated
(Fig. 8, A and B; unpublished data). Moreover, sequester-
ing NF?B in the cytosol through expression of a mutant
I?B? (I?B?M) also rendered the T4-2s anchorage depen-
dent for their survival (Fig. 8 C). Therefore, our data indi-
cate that LM-5 ligation of ?6?4 integrin likely activates
NF?B via a RAC-dependent pathway that acts upstream of
IKK?/? kinases. If true, then we reasoned that constitutive
activation of NF?B should render nonmalignant MECs
anchorage independent for growth and survival. We ad-
dressed this possibility by assaying for integrin-dependent
survival and anchorage-independent colony formation in
S-1s that overexpressed NF?B. Consistently, we found
that expressing an exogenous NF?B in S-1s led to constitu-
tive nuclear localization of p65 (Fig. 8, D and E) and per-
(A) Phase-contrast (a) and immunofluorescence microscopy images
(EGFP; a? and a??) of low (a and a?) and high (a??) magnifications of
EGFP-tagged N17 RAC showing uniform high expression of the
stable, exogenously expressed dominant-negative RAC protein in
T4-2s. Bars: (a and a?) 100 ?m; and (a??) 20 ?m. (B) Cell viability
calculated by the Live/Dead assay in control T4-2s (T4-2) and T4-2s
expressing the N17 RAC (T4 N17Rac) grown in rBM for 96 h with or
without a function-blocking mAb to ?1 integrin. (C) Immunofluores-
cence microscopy images of EGFP (FITC) detected only in T4-2s
stably expressing EGFP N19Rho. Bar, 20 ?m. (D) Cell viability
calculated by the Live/Dead assay in control T4-2s (T4-2) and T4-2s
expressing the N19Rho (T4N19Rho) grown in rBM for 96 h with or
without a function-blocking mAb to ?1 integrin. (E) Immunofluores-
cence microscopy images of c-myc (Texas red) detected only in S-1s
stably expressing a myc-tagged V12 RAC. Bar, 20 ?m. (F) Cell viability
calculated by the Live/Dead assay for S-1 controls (S-1) and S-1s
expressing a c-myc–tagged V12 constitutively active RAC (S-1 V12Rac)
grown in rBM for 96 h with or without a function-blocking mAb to
?1 integrin. (G) Soft agar assay results demonstrating that expression
of exogenous V12RAC (S-1 V12Rac) renders nonmalignant S-1s
(S-1) anchorage independent for growth and survival. Results shown
in B, D, F, and G are mean ? SEM of three experiments. *, P ? 0.05;
**, P ? 0.01.
RAC mediates anchorage-independent survival in MECs.
Breast tumors survive via ?4 integrin, RAC, and NF?B | Zahir et al. 1403
mitted S-1s to form viable colonies in soft agar (Fig. 8 G)
and to grow and survive in the absence of ?1 integrin liga-
tion (Fig. 8 F). Thus, in the absence of ECM adhesion
NF?B can sustain MEC survival.
We used paired nonmalignant (S-1) and tumor cells (T4-2)
from the HMT-3522 tumor progression model and 3D aga-
rose, collagen I, and rBM assays to investigate whether an-
chorage-independent growth and survival of mammary tu-
mors depends on autocrine LM-5 ligation of ?6?4 integrin.
We found that malignant transformation is associated with
up-regulation of ?6?4 integrin and increased LM-5 secre-
tion, and that ligation of overexpressed full-length but not a
?4?cyto, in combination with autocrine LM-5, is necessary
and sufficient to induce anchorage-independent growth and
survival in MECs, even in the absence of a polar tissue struc-
ture. Our results are consistent with the idea that MECs that
secrete sufficient quantities of LM-5 and retain ?6?4 integrin
become selected during malignant transformation because
they are able to grow and survive in the absence of exogenous
anchorage-independent survival in mammary tumors. (A)
Confocal immunofluorescence microscopy images of c-myc
(Texas red) and NF?B p65 (FITC) of S-1 controls (S-1) and
S-1 V12RAC expressing MECs (S-1 V12 Rac) grown in rBM
for 8–10 d showing high levels of nuclear NF?B p65 in the
S-1 V12 Rac MECs (arrows, nuclei as indicated by “n”) and
low to nondetectable amounts in the control S-1s (arrows).
Bar, 20 ?m. (B) Quantification of 100–200 representative
cells as shown in A, illustrating there is a significant increase
in nuclear NF?B p65 when RAC activity is elevated. (C)
Confocal immunofluorescence microscopy images of cyto-
keratin 18 (Texas red) and NF?B p65 (FITC) of T4-2 controls
(T4-2) and T4-2 revertants (T4?1) grown in rBM for 12 d
and treated with or without the Rho GTPase inhibitor Toxin
A (C. difficile; 200 ng/ml). Note the presence of detectable
nuclear p65 in the T4?1 cells (arrows) and its absence in the
Toxin A–treated structures. Bar, 20 ?m. (D) Quantification
of 100–200 representative cells from similar images shown
in C demonstrating high levels of nuclear NF?B p65 in
T4?1 structures that decrease significantly after treatment
with Toxin A. (E) Confocal immunofluorescence microscopy
images of NF?B p65 (Texas red), EGFP protein (EGFP), and
overlay of NF?B p65 and EGFP protein (yellow) in T4-2
controls (T4-2) and T4-2s expressing an EGFP-tagged tailless
?4 integrin (T4 ?4?cyto). In the absence of a cytoplasmic
?4 integrin tail, tumors fail to activate NF?B in response to
TNF-? treatment, indicated by absence of staining in the
nuclei (n) and presence of punctate staining in control nuclei
(arrow). Bar, 20 ?m. (F) Quantification of 100–200 repre-
sentative cells from similar images as shown in E illustrating
a significant increase in nuclear NF?B p65 only in T4-2s
treated with TNF-? but not in T4??4cyto cells. (G) Gel shift
showing detectable binding of a transcriptional complex
containing the NF?B p65 protein (supershift), its significant
enhancement after TNF-? treatment in T4-2s, and its absence
in T4-2s lacking the cytoplasmic tail of the ?4 integrin
(T4 ?4?cyto). (H) Cell viability calculated by the Live/Dead
assay for T4 ?4?cyto cells and T4-2s expressing both the
?4?cyto and a constitutively active RAC (T4 ?4?cyto/
V12Rac) grown in rBM for 96 h with and without a function-
blocking mAb to ?1 integrin. (I) Soft agar assay results
demonstrating that expression of exogenous V12RAC
supports anchorage-independent growth and survival of
T4 ?4?cyto cells. Results from B, D, F, H, and I are the
mean ? SEM of three to four experiments. *, P ? 0.05;
**, P ? 0.01; and ***, P ? 0.001.
?4 integrin activates NF?B via RAC to mediate
1404 The Journal of Cell Biology | Volume 163, Number 6, 2003
BM cues. Because apoptosis limits metastatic efficiency
(Wong et al., 2001), and LM-ligated ?4 integrin also sup-
ports epithelial migration and invasion (Russell et al., 2003)
and mediates immune and multi-drug resistance (Weaver et
al., 2002), our results could explain why metastatic breast tu-
mors frequently express ?4 integrin (Menard et al., 1994)
and why patients that express both BM protein and ?6?4 in-
tegrin have the worst prognosis (Tagliabue et al., 1998).
?6?4 integrin can support tumor survival through PI 3-
and AKT kinase (Bachelder et al., 1999) and keratinocyte
proliferation through ERK (Dans et al., 2001), and ?4 inte-
grin can activate NF?B through AKT and ERK (Bozinovski
et al., 2002). Yet, we found that ?6?4 integrin activates
NF?B and mediates MEC survival through RAC, and not
through AKT or ERK. One plausible explanation for the
discrepancy between our results and those published by oth-
ers is that we conducted our experiments in the context of
3D malleable gels. In contrast to cells grown as two-dimen-
sional (2D) monolayers on rigid, planar substrates, cells em-
bedded within 3D malleable gels more accurately recapitu-
late normal and malignant tissue organization and behavior
(Jacks and Weinberg, 2002). For example, MECs grown to
form tissuelike structures (acini) in 3D BM gels are able to
differentiate and optimally synthesize ? casein in response
to lactogenic hormones (Roskelley et al., 1994). Likewise,
salivary epithelial cells form acini that express cystatin only
in the context of a 3D BM gel (Royce et al., 1993), and ke-
ratinocytes recapitulate epidermal differentiation, including
fillagrin expression, most efficiently when grown as 3D organo-
typic rafts (Javaherian et al., 1998). Furthermore, MMP1
significantly enhances tumor growth in 3D, but has no ef-
fect on cell proliferation in 2D (Hotary et al., 2003); and
RAC is required for cyst polarity in MDCKs grown within
3D collagen gels, but has no effect on MDCK polarity when
cells are grown on 2D planar, rigid membranes (O’Brien et
al., 2001). Why cells behave differently when grown on a
planar, rigid substrate versus a 3D malleable gel remains an
open question. What is known is that fibroblasts do not as-
semble focal adhesions containing ?v?3 integrin and acti-
vated focal adhesion kinase in response to a 3D ECM, but
do so when plated on top of a 2D matrix (Cukierman et al.,
2001). Moreover, MECs cultured on 2D planar substrates
transiently activate MAP kinase in response to EGF, whereas
MECs grown within 3D gels to form acini do not (Wang et
al., 1998); and polarized mammary structures grown within
3D gels are recalcitrant to a diverse array of apoptotic stim-
uli, whereas MECs spread on a 2D planar substrate remain
sensitive (Weaver et al., 2002; unpublished data). Thus the
composition of integrin adhesions and integrin signaling
function appear to be differentially regulated in 2D and 3D,
implying that ?6?4 integrin may regulate epithelial survival
by different mechanisms in 2D and 3D.
The Rho GTPases, RAC and Rho, are overexpressed in tu-
mors (Fritz et al., 1999), and RAC enhances tumor invasion
in culture (Keely et al., 1997) and supports breast tumor me-
tastasis in vivo (Bouzahzah et al., 2001). We found that in
MECs, EGF stimulation of RAC depends almost entirely on
LM ligation of a full-length ?6?4 integrin. Likewise, we
found that NF?B activation also requires functional ?6?4 in-
tegrin. Because LM-5 and ?6?4 integrin are so often retained
in primary breast tumors (Tagliabue et al., 1998; Davis et al.,
2001), our results offer a plausible explanation for why RAC
and NF?B activity are frequently elevated in these same ma-
lignant tissues (Sovak et al., 1997; Fritz et al., 1999). More-
over, by establishing a functional link between RAC and
NF?B in 3D tissues, our findings could explain how RAC
supports mammary tumor growth in soft agar (Bouzahzah et
al., 2001) and why RAC supports the viability of cells actively
migrating into 3D collagen gels (Cho and Klemke, 2000). Fi-
nally, our data predict that ?6?4 integrin could drive tumor
metastasis through an alternative PI 3-kinase and Akt-inde-
pendent mechanism (Mercurio and Rabinovitz, 2001).
independent survival of MECs. (A and B) Cell viability was calculated
using the Live/Dead assay for T4-2s (A) and T4 ?4? cyto/V12RAC
(B) MECs treated with either vehicle (Control), a peptide that inhibits
nuclear translocation of NF?B SN50 (SN50), or a nonfunction-blocking
peptide SN50M (SN50M) grown in rBM for 96 h with or without a
function-blocking mAb to ?1 integrin. (C) Cell viability calculated
using the Live/Dead assay for T4-2 controls (T4-2) or T4-2s expressing
a mutant I?B? (I?B?M) grown and treated as in A. (D) Confocal
immunofluorescence microscopy images of Cytokeratin 18 (Texas
red) and NF?B p65 (FITC) in S-1 controls (S-1) and S-1s overexpressing
an exogenous NF?B p65 (S-1 p65) showing constitutive nuclear
NF?B p65 in the S-1 p65 structures (arrows, nuclei as indicated by
“n”). Bar, 20 ?m. (E) Quantification of 100–200 representative cells
assayed from images similar to D demonstrating a significant increase
in nuclear NF?B p65 in S-1s overexpressing NF?B p65 (S-1 p65). (F)
Cell viability was calculated using the Live/Dead assay for S-1 and
S-1 cells overexpressing NF?B p65 (S-1 p65) grown and treated as
described for A. (G) Soft agar assay results demonstrating overex-
pressing exogenous NFkB p65 (S-1 p65) permits S-1s (S-1) to form
colonies in soft agar. Results for A–C and E–G are the mean ? SEM
of three to five experiments. *, P ? 0.05; ***, P ? 0.001.
NF?B activation is necessary and sufficient for anchorage-
Breast tumors survive via ?4 integrin, RAC, and NF?B | Zahir et al. 1405
Current theory maintains that anoikis is circumvented
early during malignant transformation (Frisch and Ruo-
slahti, 1997) and that metastatic cells are selected thereafter
from the invasive tumor population through pressures ex-
erted by the tumor tissue microenvironment (Wouters et al.,
2003). Yet, tumor metastasis can occur early during cancer
(Wasserberg et al., 2002); metastatic cells have been found
in the bone marrow of patients with early stage tumors (Me-
nard et al., 1994), and tumor cells do circulate in the blood
of patients with benign disease (Hardingham et al., 2000).
Metastatic tumors frequently express integrins such as ?v,
?5, ?6, and ?4, and often secrete ECM proteins including
collagen IV, LM-5, and fibronectin (Davis et al., 2001; Ioa-
chim et al., 2002). Tumor metastasis and extravasation are
facilitated by integrin–ECM interactions (Clezardin, 1998).
Therefore, it is plausible that apoptosis-resistant metastatic
tumors arise early during malignancy through selection of
transformed cells that express ECM proteins and retain inte-
grins that support migration, invasion, and survival. Because
we show that malignant transformation is linked to auto-
crine LM-5, that LM-5 supports cell survival by inducing
?6?4 integrin–RAC–NF?B signaling, and that LM-5–
ligated ?6?4 integrin and RAC support epithelial motility
and invasion (Russell et al., 2003), our data underscore the
feasibility of this concept.
Materials and methods
We used commercial EHS matrix (Matrigel; Collaborative Research) for the
rBM assays; Vitrogen 100 (bovine skin collagen I; Celtrix Laboratories), 3
mg/ml and 10 ?g/ml of affinity-purified LM-5 (Russell et al., 2003) for coat-
ing culture dishes; and 0.3% Cellagen Solution AC-5 (ICN Biomedicals) for
the 3D collagen I assays. Primary antibodies were as follows: LM-5, rabbit
sera pKa1, and clone BM165 (Russell et al., 2003); ?6 integrin, clone
GoH3 (BD Biosciences); ?1 integrin, clones AIIB2 (provided by C. Dam-
sky, University of California, San Francisco, San Francisco, CA), and TS2/
16 (ATCC); ?4 integrin, rabbit sera, and clones 3E1, ASC-3, and ASC-8;
and ?2 integrin, clone 10G11 (all from Chemicon International); I?B?/
MAD-3, clone 25, and NF?B p65, rabbit sera (Santa Cruz Biotechnology,
Inc.) and clone 20 (BD Biosciences); cytokeratin 18, clone RCK106 (BD
Biosciences); RAC1, clone 102 (BD Biosciences); c-myc, clone 9E10 (On-
cogene Research Products), and AKT and Phospho-ser472/473/474-AKT;
ERK1, rabbit sera (BD Biosciences), phosphoERK1/2 (Thr202/Tyr204), rab-
bit sera (New England BioLabs, Inc.); activated caspase 3, rabbit sera (Cell
Signaling), and HA.11, clone 16B12 (Babco). Secondary antibodies were
as follows: horseradish peroxidase, and biotinylated mouse IgG (Vector
Laboratories); FITC, and Texas red–conjugated and nonconjugated anti–
mouse, anti–rat, and anti–rabbit goat polyclonal antibodies and nonspe-
cific rat and mouse IgGs (Jackson ImmunoResearch Laboratories). Re-
agents were as follows: NF?B SN50, active cell-permeable inhibitor
peptide (50 ?M in PBS), NF?B SN50M, inactive cell-permeable control
peptide (50 ?M in PBS); the EGFR-specific tyrosine kinase inhibitor Tyr-
phostin AG 1478 (160 ?M in DMSO), and the Rho GTPase inhibitor toxin
A Clostridium difficile (10 mM in DMSO; Calbiochem); the MEK1 inhibitor
PD98059 (50 ?M in DMSO); and the PI 3-kinase inhibitor LY 294002 (50
?M in ethanol; BIOMOL Research Laboratories, Inc.).
The HMT-3522 MECs were grown in 2D and embedded (0.5–0.8 ? 106
cells/ml) within ECM gels and phenotypic reversion of T4-2s using ?1 inte-
grin mAb AIIB2 or Tyrphostin AG 1478 as described previously (Wang et
Cell adhesion was assessed using a fluorescence attachment assay. In
brief, plates coated with LM-5 or rBM (100 ?g/ml) were blocked (1 h;
0.1% BSA), incubated (60 min, 37?C), washed (3? PBS), incubated with 4
?M calcein (20 min, RT), and quantified using a fluorescence plate reader
(model Fluoroskan Ascent Fl; LabSystems).
Anchorage-independent growth was assessed using a soft agar assay
(Wang et al., 1998). In brief, 20,000 cells were plated in 1 ml DME/Ham’s
F12 containing 0.7% agarose, overlaid with 1 ml of 1% agarose, and 40-
?m colonies were scored positive after 21 d.
To inhibit integrin function or LM-5 binding, cells were incubated with
mAbs against ?1 integrin, clone AIIB2 (1:25–1:100 ascites/ml ECM); ?4 in-
tegrin, clones ASC-3 or ASC-8 (4–16 ?g IgG/ml ECM); LM-5, clone BM165
(10 ?g IgG/ml ECM); or IgG isotype-matched control mAb (4–16 ?g IgG/
ml ECM) at the time of embedment. To inhibit NF?B nuclear translocation,
the active inhibitor NF?B SN50 or the inactive analogue NF?B SN50M
was added directly to the media.
Cells were directly fixed using 2–4% PFA or 100% methanol, and samples
were incubated with primary mAbs, followed by either FITC- or Texas red–
conjugated secondary antibodies. Nuclei were counterstained with DAPI
(Sigma-Aldrich). Cells were either visualized using a scanning confocal la-
ser (model 2000-MP; Bio-Rad Laboratories) attached to a fluorescence mi-
croscope (model Eclipse TE-300 [Nikon] or model MDIRBE [Leica]). Con-
focal images were recorded at 120? and conventional images were
recorded at 40–60?.
Apoptosis was assayed by the Live/Dead Assay (Molecular Probes) or by
detection of internucleosomal DNA fragmentation in fixed cells using an
in situ TUNEL assay (Boehringer) or via immunodetection of activated cas-
pase 3. Percent death was calculated as cells positive for ethidium bro-
mide expressed as a percentage of the total number of live cells scored
positive by calcein staining (FITC). The apoptotic labeling index was cal-
culated as the percentage of total cells positive for FITC-labeled 3?OH
DNA ends, and percent apoptosis was determined as the percentage of to-
tal cells positive for activated caspase 3. The minimum number of cells
scored was 200–400 per experimental condition. Cell death by apoptosis
was confirmed by showing that DNA cleavage or caspase 3 activity could
be inhibited by prior treatment with the caspase inhibitors YVAD CHO or
DEVD-CHO (1 ?M; BIOMOL Research Laboratories, Inc.).
Full-length ?4 pRK-5 (provided by F. Giancotti, Memorial Sloan-Kettering
Cancer Center, New York, NY) was used directly. The 2,710-bp EcoRI–
BglII fragment from the ?4pRK-5 construct was ligated with the EcoRI–
BamHI vector fragment of pEGFP-N2, and an EcoRI–NotI fragment con-
taining the ?4 integrin EGFP fusion was subcloned into an EcoRI–NotI
vector fragment of Hermes HRS puro-GUS (provided by H. Blau, Stanford
Medical Center, Stanford, CA). Myc-tagged V12RAC1 (provided by A.
Hall, University College, London, UK) was cloned as an EcoRI fragment
into LZRS-IRES-blasticidin; and N17RAC1 and N19RhoA (provided by E.
Butcher, Stanford Medical Center) were cloned into the EGFP fusion vector
EGFP-C1 (CLONTECH Laboratories, Inc.), and excised and recloned into
LZRS-IRES-blasticidin by PCR using the EcoRI tailed primer GTPaseF, 5?.
I?B?M and p65 cloned into PLZRS (provided by P. Khavari, Stanford Med-
ical Center) were used directly. The BglII–BamHI fragment containing HA-
tagged dominant-negative AKT (K179M) and the HindIII–EcoRI fragment
containing the myristoylated HA-tagged AKT (provided by P. Tsichlis, Tufts
University, Boston, MA) were subcloned into pLZRS.
Gene expression manipulations
Amphotrophic retrovirus was produced in either modified 293 cells or in
Phoenix ampho cells (provided by G. Nolan, Stanford Medical Center),
and MECs were spin infected and selected using blasticidin. MECs were
transfected with full-length ?4 pRK-5 and pcDNA 3.1 plasmid vector DNA
or vector plasmid alone using LipofectAMINE (GIBCO BRL), and selected
using G418. S-1 ?4 pRK-5–transfected cells were enriched for increased
membrane localized ?4 integrin through differential adhesion to LM-5,
and increased ?4 integrin levels were verified by FACS® analysis.
Electrophoretic mobility shift assay
To prepare nuclear extracts, cells were washed (1? PBS) and homoge-
nized in nuclear isolation buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 1
1406 The Journal of Cell Biology | Volume 163, Number 6, 2003
mM EDTA, 1 mM EGTA, 1mM DTT, and 1 mM Pefabloc SC) with an addi-
tion of IGEPAL to 0.5%. After incubation (10 min, 4?C), nuclei were iso-
lated by centrifugation (1 min, 14,000 rpm, 4?C) and nuclear extracts were
prepared by homogenization and incubation in nuclear extraction buffer
(20 mM Hepes, pH 7.9, 420 mM KCl, 1.5 mM MgCl2, 20% glycerol, 0.5
mM DTT, 1 mM Pefabloc SC, and 10 ?g/ml leupeptin), followed by cen-
trifugation (15 min, 14,000 rpm, 4?C). Equal amounts of nuclear protein
were used in the EMSA reaction with the NF?B consensus oligonucleotide
sequence (5?-AGT TGA GGG GAC TTT CCC AGG C-3?). 32P-Labeled oli-
gonucleotide (150,000 cpm) was incubated with 5 ?g of nuclear extract
and gel shift binding buffer (10 min, RT; Promega Gel Shift Assay System).
p65 rabbit antisera were added after the binding reaction, and the mixture
was reincubated (20 min, RT). Specificity of binding was tested using com-
petition analyses in which 10-fold molar excess of nonlabeled oligonucle-
otide sequence was added to a binding reaction. Complexes were resolved
in 4.5% polyacrylamide gels (TE buffer: 90 mM Tris, 90 mM boric acid,
and 2 mM EDTA, pH 8.0).
Cells were isolated, nonspecific binding was blocked (60 min Dulbecco’s
PBS, 0.1% BSA) and incubated with saturating concentrations of primary
mAb (1 h), washed three times with Dulbecco’s PBS, and labeled with
FITC-conjugated goat immunoglobulin. Stained cells were washed three
times with Dulbecco’s PBS and immediately analyzed on a FACScan™
(Becton Dickinson). All manipulations were conducted at 4?C.
Cells were lysed (RIPA buffer: 50 mM Tris-HCl, pH 7.4, 150 mM sodium
chloride, 1% NP-40, 0.5% deoxycholate, 0.2% SDS containing 20 mM so-
dium fluoride, and 1 mM sodium orthovanadate, and a cocktail of pro-
tease inhibitors), and equal amounts of protein were separated on reducing
SDS-PAGE gels, immunoblotted, and detected with an ECL-Plus system
(Amersham Biosciences). To assay for differences in total secreted LM-5,
LM-5 was immunoprecipitated from conditioned media, and protein from
equal cells was separated on SDS–polyacrylamide gels, immunoblotted,
and detected as above.
Cells were treated with vehicle or 20 ng/ml EGF and incubated for indicated
times, washed (2? PBS), and extracted (G protein buffer: 25 mM Hepes, pH
7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 10%
glycerol, 1 mM Pefabloc SC, 10 ?g/ml leupeptin, 10 ?g/ml aproptinin, 1
mM sodium orthovanadate, and 1 mM sodium fluoride; 5–10 min). Lysates
were centrifuged (10 min, 14,000 rpm), and supernatants were mixed with
GST-PBD and incubated with glutathione-Sepharose beads (Amersham Bio-
sciences; 60 min). Lysates were washed (3? lysis buffer), and bound protein
was eluted with Laemmli buffer and separated on a 12% SDS–polyacryl-
amide gel. Active RAC was detected by immunoblotting with anti-RAC anti-
body, and specific activity was calculated by normalizing densitometric val-
ues of PAK-associated RAC to total RAC and E-cadherin. Purified GST-PBD,
encoding amino acids 70–117 of PAK1, fused to GST (provided by J. Cher-
noff, Fox Chase Cancer Center, Philadelphia, PA).
We thank C. Damsky for the AIIB2 mAb; Drs. P. Khavari, P. Tsichlis, A.
Hall, E. Butcher, and F. Giancotti for cDNA clones; J. Chernoff for GST hu-
man PAK1 cDNA; G. Nolan for the Phoenix ampho cells; and Z. Werb and
N. Boudreau for helpful comments.
This work was supported by National Cancer Institute grant CA 78731
and Department of Defense grant DAMD17-01-1-0368 to V.M. Weaver
and grant DAMD17-01-1-0367 to J.N. Lakins; National Institutes of Health
(NIH) grant P01 AR44-012 to A. Russell and A.P. Marinkovich; and NIH
grant T32 HL07954-03 to N. Zahir.
Submitted: 5 February 2003
Accepted: 27 October 2003
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