In vivo selection for metastasis promoting genes
in the mouse
Kiranmai Gumireddy*, Fangxian Sun†, Andres J. Klein-Szanto‡, Jonathan M. Gibbins§, Phyllis A. Gimotty¶,
Aleister J. Saunders?, Peter G. Schultz†**††, and Qihong Huang*††
*The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104;†Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive,
San Diego, CA 92121;‡Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497;§School of Animal and Microbial Sciences, University of
Reading, Whiteknights, Reading RG6 6AJ, United Kingdom;¶Department of Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia,
PA 19104;?Department of Bioscience and Biotechnology, Drexel University, 3141 Chestnut Street, 316 Stratton Hall, Philadelphia, PA 19104; and
**The Scripps Research Institute, 10550 North Torrey Pines Road, SR 202, La Jolla, CA 92037
Contributed by Peter G. Schultz, February 12, 2007 (sent for review December 1, 2006)
Here, we report the identification of a metastasis promoting factor
by a forward genetic screen in mice. A retroviral cDNA library was
introduced into the nonmetastatic cancer cell line 168FARN, which
was then orthotopically transplanted into mouse mammary fat
pads, followed by selection for cells that metastasize to the lung.
The genes encoding the disulfide isomerase ERp5 and ?-catenin
were found to promote breast cancer invasion and metastasis.
Disulfide isomerases (thiol isomerases), which catalyze disulfide
bond formation, reduction, and isomerization, have not previously
been implicated in cancer cell signaling and tumor metastasis.
Overexpression of ERp5 promotes both in vitro migration and
invasion and in vivo metastasis of breast cancer cells. These effects
were shown to involve activation of ErbB2 and phosphoinositide
3-kinase (PI3K) pathways through dimerization of ErbB2. Activa-
tion of ErbB2 and PI3K subsequently stimulates RhoA and ?-
catenin, which mediate the migration and invasion of tumor cells.
Inhibition of ErbB2 and PI3K reverses the phenotypes induced by
ERp5. Finally, ERp5 was shown to be up-regulated in human
surgical samples of invasive breast cancers. These data identify a
link between disulfide isomerases and tumor development, and
provide a mechanism that modulates ErbB2 and PI3K signaling in
the promotion of cancer progression.
breast cancer invasion and metastasis ? disulfide isomerase ERp5 ?
ErbB2/phosphoinositide 3-kinase ? forward genetic screen ?
orthotopic animal model
tasis occurs by a multistep process requiring the coordinated action
of many genes (2–5): the escape of cancer cells from the primary
tumor and entry into the blood stream (intravasation), survival in
the circulation, exit from capillaries into surrounding tissues at a
new site (extravasation), initiation of growth to form micrometas-
tases, and development of new blood vessels to form secondary
tumors (2–5). Although a number of mechanisms have been
identified that induce tumor cell migration and invasion, including
the activation of cell surface receptors and small GTPases of the
Rho family (6–13), the systematic identification and characteriza-
tion of additional genes that promote or suppress tumor invasion
process and ultimately provide new therapeutic targets for the
treatment of cancer.
Forward genetic screens provide an unbiased approach to the
identification of genes that contribute to a phenotype of interest
for both gain-of-function and loss-of-function cell-based screens at
the genome-wide level (17–21). However, due to the complexity of
metastasis, no cell-based screen accurately recapitulates the entire
of metastatic tumors in secondary organs. Thus, the application of
animal models of metastasis to such genetic screens should facili-
tate the systematic identification of genes that play critical roles in
etastasis remains one of the most poorly understood pro-
cesses in cancer biology despite extensive study (1). Metas-
this process. In this study, an in vivo selection system in the mouse
was used to identify genes from a large cDNA library that com-
plement a cell line defective in the early steps of metastasis. Two
gene products, ?-catenin and the disulfide isomerase ERp5, were
model. Additional experiments demonstrated that ERp5 promotes
breast cancer cell migration and invasion through activation of the
EGF receptor ErbB2/PI3K pathway and downstream signaling
molecules, including Akt and RhoA. In addition, ERp5 was found
to be up-regulated in invasive clinical breast cancer samples. These
invasion and metastasis and provide an additional mechanism for
modulation of ErbB2/PI3K signaling.
Results and Discussion
A Forward Genetic Screen in an Orthotopic Mouse Model for Metas-
tasis Promoting Genes. Ourgeneticscreenformetastasispromoting
genes is based on orthotopic transplantation of tumor cells infected
with a retroviral cDNA library into the mouse mammary fat pad,
transplantation mouse model was used (instead of a tail vein
injection model) because transplanted tumor cells in the mammary
fat pad must complete all of the steps of metastasis to spread to the
lung. 168FARN cells, originally isolated from a single mouse
mammary tumor, which arose spontaneously in a wild type
BALB/cJ mouse (22), were chosen for the initial screen. 168FARN
cells are able to grow at the primary site but are not able to develop
metastatic nodules when transplanted into the mammary fat pad
(Fig. 1B). However, 168FARN cells develop lung metastasis when
injected directly into the blood stream through the tail vein (Fig.
(i.e., tumor migration and invasion), but have full metastatic
the early steps of tumor spread. Any gene that can complement the
metastatic defects in this cell line should cause cells to metastasize
from the mammary fat pad to the lung, which serves as a positive
To carry out the screen, a retroviral cDNA library consisting of
cDNAs prepared from day 13.5 embryonic stage mice and ex-
of cDNAs into the host genome after retroviral infection makes it
tools; K.G., F.S., A.J.K.-S., and Q.H. analyzed data; and K.G., P.G.S., and Q.H. wrote the
The authors declare no conflict of interest.
Abbreviations: PI3K, phosphoinositide 3-kinase; DTNB, 5,5?-dithiobis(2-nitrobenzoic acid).
††To whom correspondence may be addressed. E-mail: email@example.com or qhuang@
© 2007 by The National Academy of Sciences of the USA
April 17, 2007 ?
vol. 104 ?
possible to easily retrieve cDNAs from the positively selected cells
by PCR (23). 168FARN cells (1 ? 106) that contain the cDNA
library were transplanted into the mouse mammary fat pad (Fig.
1A). A proof-of-concept experiment using five BALB/cJ mice was
performed. Of these five mice, two subsequently developed lung
metastasis in 7 weeks (Fig. 1C). Genes encoding ?-catenin and
ERp5 were retrieved from the metastatic cells of these mice.
?-Catenin is a major downstream effector of the Wnt pathway (24,
25) that impairs epithelial cell differentiation and induces the
epithelial-mesenchymal transition (26–29). In addition, nuclear
?-catenin activity is up-regulated in various types of aggressive
cancers (30–32). The isolation of a gene that is known to play an
important role in tumor metastasis validates the ability of this
approach to identify bona fide metastasis promoting genes.
ERp5 is a member of the thiol isomerase family which is
responsible for the formation of native disulfide bonds in cell
surface and secreted proteins (33–37). These enzymes ensure the
correct pairing of cysteine (Cys) residues in proteins by catalyzing
the formation and rearrangement of disulfide bonds. This process
ensures the correct folding of their substrates into native confor-
to their localization in the endoplasmic reticulum, some thiol
isomerases are also located on the cell surface where they function
shown that ERp5 is recruited to the cell surface and plays a major
role in platelet aggregation in response to platelet agonists (39).
Despite extensive studies of the thiol isomerase family, little is
known regarding their roles in cellular signaling pathways and
To validate the metastasis-promoting activity of ERp5, the gene
was cloned into a retroviral vector behind the LTR promoter and
introduced into 168FARN cells to generate a cell line that stably
expresses ERp5. Overexpression of ERp5 was confirmed by im-
munoblot with an ERp5 antibody (data not shown). These cells
were then transplanted into the mammary fat pad using the same
procedure as in the initial screen. Lung metastasis nodules devel-
oped after transplantation, demonstrating that ERp5 promotes
tumor metastasis in vivo, whereas 168FARN cells containing a
control vector do not cause metastasis (Fig. 1D).
metastasis-promoting genes in this proof-of-concept study. The
cDNA library that was used had a titer of 107to 108cfu, which is
much larger than the coverage of cDNAs that can be obtained with
five mice. Many genes, especially genes with low copy numbers,
might be found using larger numbers of mice. The other possibility
is that the cDNA library was prepared from day 13.5 embryonic
mice and contains only genes that are expressed during this stage.
An ideal cDNA library for this type of study is one that contains all
genes in the genome, where each gene has equal representation in
the library. Such a library is now being generated for future studies.
ERp5 Promotes Tumor Cell Migration and Invasion in Vitro. Because
168FARN cells are defective in the early steps of tumor metastasis,
it is likely that ERp5 plays a role in initial tumor migration and
shows no metastasis in secondary organs (B Lower). 168FARN cells are able to form lung metastasis after tail vein injection (1 ? 106cells) (B Upper Right). (C and
D) Screening and validation: Tumor cell growth in the primary site after injection of 168FARN cells (1 ? 106cells) containing a cDNA library in mammary fat pad.
(C) Two of five mice developed lung metastasis. Transplantation of 168FARN cells stably overexpressing ERp5 in mammary fat pad leads to lung metastasis. (D)
Mice were imaged 6–8 weeks after transplantation.
Identification of ERp5 as a metastasis-promoting gene. (A) The scheme for the forward genetic screen. (B) Model establishment: 168FARN tumor cell
Gumireddy et al.
April 17, 2007 ?
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invasion into neighboring tissues. The effect of ERp5 on these
initial steps was therefore evaluated in a transwell cell migration
its expression driven by the LTR promoter and was subsequently
introduced into human breast cancer cell lines MCF7 and MDA-
MB-436 to establish cells stably expressing ERp5. Overexpression
of ERp5 in both cell lines was confirmed by immunoblot with an
ERp5 antibody (data not shown). Overexpression of ERp5 in
MCF7 cells results in a migratory phenotype, whereas cells con-
taining a control vector are nonmigratory. In addition, MDA-MB-
in the number of migrated cells compared with the cells containing
a control vector (Fig. 2A). Similar results were obtained in an
invasion assay in which cells must penetrate Matrigel during mi-
gration. The phenotype of MCF7 cells that overexpress ERp5
changes from noninvasive to invasive, whereas MDA-MB-436 cells
overexpressing ERp5 display more than a 60% increase in the
number of invasive cells (Fig. 2B). Similar results were obtained in
168FARN cells as well. Taken together, these results demonstrate
that ERp5 promotes tumor cell migration and invasion.
It has been shown that some disulfide isomerases regulate cell
surface protein function through disulfide exchange at the cell
surface (38–41). The role of disulfide exchange in cell migration
and invasion was examined by treatment of cells with 5,5?-
dithiobis(2-nitrobenzoic acid) (DTNB), an activated disulfide
which reacts with free thiols and thereby inhibits cell surface thiol
of MDA-MB-436 parent and ERp5-overexpressing cells by 40%
and 80%, respectively. Furthermore, migration of MCF7 cells that
overexpress ERp5 is reduced by ?60% upon treatment with 1 mM
DTNB (compared with DMSO-treated cells) (Fig. 2C). Similarly,
1 mM DTNB inhibits invasion of MDA-MB-436 parent and ERp5
overexpressing cells by 30% and 80%, respectively, and inhibits
invasion of MCF7 cells overexpressing ERp5 by ?80% (Fig. 2D).
effect of inhibiting cell surface disulfide exchange is more promi-
nent in cells that overexpress ERp5, which may reflect a dominant
effect of thiol exchange on cell migration and invasion when ERp5
is expressed at high levels. These results not only show that cell
surface disulfide exchange is critical for the activity of ERp5 on cell
exchange may be beneficial in the treatment of tumor metastasis.
Activation of the ErbB2 Pathway Is Required for the Metastatic
Activity of ERp5. Because ERp5 is known to modulate cell surface
receptors (38, 40), we next examined its effect on candidate genes
known to play an important role in breast cancer development.
ErbB2 (HER2/NEU) is one such gene. It is a member of subclass I
of the receptor tyrosine kinase superfamily (42), which when
activated leads to the stimulation of many cell signaling pathways
including PI3K-AKT pathways, the mitogen-activated protein ki-
nase pathway, and the SRC tyrosine kinase pathway (43, 44). The
expression of ErbB2 is altered in many epithelial tumors such as
breast, ovarian, gastric, and non-small-cell lung cancers (45–48).
ErbB2 expression has also been shown to be inversely correlated
with the prognosis of breast cancer, indicating an important role in
tumor metastasis (45, 49, 50).
Overexpression of ERp5 did not affect ErbB2 expression levels
in MCF7 and MDA-MB-436 cells as determined by immunoblot
analysis (Fig. 3A). However, overexpression of ERp5 led to acti-
vation of ErbB2 as determined by immunoprecipitation and West-
ern blot analysis of cell extracts from MCF7 and MDA-MB-436
cells. The level of ErbB2 phosphorylation is significantly higher in
MCF7 and MDA-MB-436 cells expressing ERp5 than in cells with
an empty vector (Fig. 3B). Similar results were obtained in
168FARN cells as well. ErbB2 triggers signal transduction by
ligand-dependent heterodimerization or ligand-independent ho-
expression induces dimerization of ErbB2 in both MCF7 and
MDA-MB-436 cells (Fig. 3C). To determine whether ErbB2 is
required for the activity of ERp5 in tumor cell migration and
invasion, a short hairpin RNA (shRNA) construct was used to
that overexpress ERp5. Western blot analysis demonstrated that
the shRNA is effective in suppressing ErbB2 when compared with
a control shRNA (Fig. 3D). Migration and invasion assays were
then performed to determine whether ERp5 activity depends on
ErbB2 expression. The number of migrated MCF7 and MDA-MB-
436 cells that express both ERp5 and the shRNA against ErbB2 is
reduced by ?90% and 85%, respectively, in comparison to cells
expressing ERp5 and a control shRNA (Fig. 3D). Similar results
were obtained in the invasion assay: invasive MCF7 and MDA-
436 and MCF7 cells were serum-starved for 24 h before the transwell assay.
Complete medium was added to the bottom wells of the transwell chambers.
Cells that migrated to the lower surface of the filters were fixed and counted
in human breast cancer MDA-MB-436 cells causes 100% more migrated cells
(A) and 60% more invaded cells (B) in a transwell assay compared with these
cells with a control vector. The expression of ERp5 in human breast cancer
exchange blocks cell migration and invasion. Treatment of cells with 1 mM
DTNB reduces migration of MDA-MB-436 cells by 40%, reduces migration of
MDA-MB-436 cells overexpressing ERp5 by ?80%, and reduces migration of
MCF7 cells overexpressing ERp5 by ?60% compared with DMSO treatment as
a control (C). DTNB (1 mM) has a similar effect on cell invasion: MDA-MB-436
cell invasion is inhibited by 30%; invasion by MDA-MB-436 cells overexpress-
ing ERp5 is inhibited by 80%; and invasion by MCF7 cells overexpressing ERp5
is inhibited by ?80% (D). DTNB (1 mM) has no effect on cell growth (E).
ERp5 promotes tumor cell migration and invasion in vitro. MDA-MB-
www.pnas.org?cgi?doi?10.1073?pnas.0701145104 Gumireddy et al.
MB-436 cells that express both ERp5 and the shRNA against
ErbB2 are reduced by ?95% compared with cells that express
ERp5 and a control shRNA (Fig. 3D). Interestingly, the effect of
ErbB2 knockdown on migration and invasion in MCF7 and MDA-
MB-436 cells overexpressing ERp5 is more prominent than in cells
containing the control vector (Fig. 3 C and D). This may again be
results demonstrate that activation of the ErbB2 pathway is re-
quired for the effects of ERp5 on tumor migration and invasion.
ERp5 Activates PI3K and Its Downstream Signaling Molecules RhoA
and ?-Catenin. Activation of ErbB signaling results in a number of
downstream events, including activation of the PI3K signaling
cascade. The effects of ERp5 on PI3K activity were therefore
examined by measuring the phosphorylation status of the p85
subunit of PI3K, which correlates with PI3K kinase activity in vivo
(53, 54). p85 phosphorylation was determined by immunoprecipi-
tation using a phosphotyrosine antibody followed by Western blot
analysis by using an antibody specific for p85. The level of active
than in those cells containing a control vector, whereas the total
amount of PI3K is similar in these samples, demonstrating that
and wortmannin were then used to examine whether PI3K is
required for the effects of ERp5 on migration and invasion. As
shown in Fig. 4B, the addition of 10 ?M LY294002 reduces
migration of MCF7 and MDA-MB-436 cells that stably express
ERp5 by ?90%; likewise, treatment with 0.1 ?M wortmannin
reduces migration by ?85% in these cells (Fig. 4B) (these PI3K
have minimal migration and invasion capacity in the absence of
ERp5 overexpression). Both compounds have no effect on cell
growth at the concentrations that were used in the experiments
(data not shown). LY294002 also reduced invasion of MCF7 and
MDA-MB-436 cells that stably express ERp5 by ?85% and 95%,
respectively; wortmannin had similar effects (Fig. 4B). Taken
together, these results strongly suggest that the PI3K pathway is
and invasion by ERp5.
The activation of downstream signaling molecules in the PI3K
pathway by ERp5 was examined next. RhoA is one of the down-
cell migration (55). Affinity precipitation of active RhoA by Rho-
tekin binding domain beads in the cell extracts of MCF7 and
MDA-MB-436 cells overexpressing ERp5 revealed higher expres-
sion levels of the active form of RhoA when compared with cells
containing a control vector; the total RhoA levels are similar in the
target of activated PI3K is the serine-threonine kinase Akt, which
is activated when it is phosphorylated at Thr308and Ser473(56, 57).
As shown in Fig. 4C, the total protein levels of Akt are similar in
cells stably expressing either ERp5 or a control vector. However,
308 and 473 are significantly increased in cells expressing ERp5
(Fig. 4C). To further explore the downstream consequences of
PI3K/Akt activation, we examined the activation of ?-catenin by
Akt. Western blot analysis confirmed that the expression levels of
?-catenin are significantly increased in MCF7 and MDA-MB-436
cells stably expressing ERp5 (Fig. 4C). These results suggest that
activation of the PI3K pathway is responsible for tumor cell
migration and invasion induced by ERp5 (Scheme 1).
ERp5 Is Up-Regulated in Invasive Clinical Breast Cancer Samples.
Finally, tissue microarrays were used to examine the expression of
ERp5 in clinical breast cancer samples at various stages. Immuno-
staining of ERp5 showed that normal breast ducts as well as early
samples) exhibited little or no ERp5 expression. However, ERp5
were stained exclusively in the cytoplasm. None of the normal
breast samples and only 4.3% of the T1N0 samples received 2?
(A) The expression level of total ErbB2
does not change after ERp5 overexpres-
sion in human breast cancer MDA-MB-436
and MCF7 cells. (B) Immunoprecipitation
of phosphorylated ErbB2 demonstrates
ErbB2 is activated by ERp5 overexpression
in MDA-MB-436 and MCF7 cells. (C) Immu-
noblots under nonreducing condition
show that ERP5 promotes dimerization of
ErbB2 in MDA-MB-436 and MCF7 cells. (D)
Knockdown of ErbB2 by a specific shRNA
reduces the effect of ERp5 on migration
and invasion in MDA-MB-436 and MCF7
ERp5 activates ErbB2 pathway.
The molecular mechanisms of ERp5 in promoting tumor invasion
Gumireddy et al.
April 17, 2007 ?
vol. 104 ?
no. 16 ?
samples scored 2? or higher (Fig. 5B), demonstrating that ERP5
is up-regulated in more invasive breast cancer samples. The P value
of Fisher’s exact test is 0.0034, indicating that there is indeed an
association between the staining results and disease progression.
These results further support the role of ERp5 in tumor progres-
sion. Interestingly, macrophages were strongly stained and endo-
thelial cells were also moderately stained with ERp5 antibody,
The immunostaining results with clinical breast cancers suggest
that tumors with no ErbB2 amplification can still be aggressive due
to the activation of ErbB2 by mechanisms other than gene ampli-
fication. Therefore, the activation of the ErbB2 pathway, rather
than the amplification or overexpression of ErbB2, may be another
indication of the need for chemotherapies targeting ErbB2. The
and PI3K inhibition in migration and invasion assays (Figs. 3D and
4B) suggests that chemotherapy targeting ErbB2 and PI3K may be
effective in patients in which ERp5 expression is up-regulated.
In summary, we have applied a novel gain-of-function in vivo
in the early steps of metastasis. ERp5 promotes tumor cell migra-
tion and invasion by activating the ErbB2/PI3K signaling pathway
the amplification of ErbB2/PI3K signaling by a disulfide isomerase.
The activation of ErbB2 by ERp5 leads to the activation of PI3K
and, subsequently, RhoA and ?-catenin, which have been shown to
play important roles in tumor invasion and metastasis (Scheme 1).
Together with the finding that ?-catenin was also identified in this
of ErbB2/PI3K pathway is critical in breast cancer metastasis,
especially in the early stages of the process. Besides its location in
the endoplasmic reticulum, ERp5 also exists on the cell surface
based on a previous publication (39) and our own observation
(unpublished data). Because the majority of cell surface proteins
contains at least one disulfide bond, these proteins may require
disulfide exchange may play an important role in the activation and
regulation of cell signaling pathways (58). In either case, the
selective inhibition of cell surface thiols may serve as a potential
therapeutic approach to the suppression of tumor progression and
Materials and Methods
Plasmids and Reagents.HumanandmouseERp5geneswerecloned
into the pBabe-puromycin plasmid and expressed by the LTR
promoter. shRNA against ErbB2 and control shRNA were pur-
chased from Open Biosystems (Huntsville, AL). Anti-ERp5 anti-
body was prepared as previously described (39). Anti-human Akt,
phospho-Akt-(Ser-473), phospho-Akt-(Thr-308), and PI3K inhibi-
tor Ly294002 were purchased from Cell Signaling Technology
(Beverly, MA); ?-catenin was purchased from BD Transduction
Laboratories (San Jose, CA); and ErbB2, phosphorylated tyrosine,
clone 4G10, PI3-kinase p85, PI3K inhibitor wortmannin, and the
RhoA activation assay kit were purchased from Upstate Cell
Signaling Solutions (Lake Placid, NY).
Cell Culture. The mouse breast cancer cell line 168FARN and
human breast cancer cell line MCF7 and MDA-MB-436 were
with 5% CO2.
Recombinant Retroviruses and Viral Infection. Virus production and
infection were performed as previously described (21).
Immunoblotting and Immunochemistry. Immunoblotting and immu-
noprecipitation were performed as previously described (21). Rho
activity was measured by affinity precipitation of GTP-Rho with
lated PI3K in MDA-MB-436 and MCF7 cells demonstrates that ERp5 overexpres-
of ERp5 in the migration and invasion assay in MDA-MB-436 and MCF7 cells. (C)
Stable ERp5 overexpression in MDA-MB-436 and MCF7 cells leads to the activa-
tion of RhoA and Akt but has no effect on the total protein expression level of
these two proteins. The activation of Akt subsequently up-regulates ?-catenin.
ERp5 activates PI3K pathway. (A) Immunoprecipitation of phosphory-
array containing normal breast tissues and breast ductal carcinoma at various
the strongest staining. ERp5 expression is elevated in T2N0 and T2N1 compared
with normal breast duct and T1N0 tumors. (B) The percentage of tumor samples
on the array with different scores was tabulated. The percentage of tumor
and T2N1 stage, respectively, compared with 0% in normal breast ductal struc-
ture and 4.3% in T1N0 tumors.
www.pnas.org?cgi?doi?10.1073?pnas.0701145104Gumireddy et al.
Rhotekin-agarose beads (Upstate Biotechnology, Lake Placid, Download full-text
NY) according to the manufacturer’s instructions.
Tissue microarrays of formalin-fixed, paraffin-embedded sam-
ples of human breast epithelium representing normal as well as
different stages of tumor progression were prepared at Fox Chase
Cancer Center. A total of 16 normal breast areas, 23 T1N0, 23
represented by a total of 172 cores were used in the tissue
microarrays. Immunostaining was performed as previously de-
was scored according to the stain intensity from 0 (no stain), 1
(marginal to moderate stain), 2 (moderate to intense stain), and 3
(strong intense stain).
Invasion and Migration Assays. Matrigel invasion assays were per-
Costar). In some experiments, cells were preincubated with the
PI3-kinase inhibitors LY294002 (100 ?M), wortmannin (100 nM),
DTNB (1 mM), or control medium for 30 min. After detachment
with trypsin, cells were washed with PBS and resuspended in
serum-free medium, and 250-?l cell suspension (2 ? 105cells/ml)
was added to the upper chamber. DTNB (1 mM) was added to the
cell suspension. Images of three different ?10 fields were captured
from each membrane, and the number of invading cells was
counted. The mean of triplicate assays for each experimental
condition was used. For migration assays, the same procedure was
performed except that transwell chambers were not coated with
Functional Screen in an Orthotopic Animal Model. To establish
luciferase positive cancer cells, the luciferase gene from pGL3
(Promega, San Luis Obispo, CA) was cloned into pBabe-
puromycin. 168FARN cells were infected with retroviruses con-
taining luciferase to establish 168FARN-Luc cells that stably ex-
press luciferase. To establish the animal model for the forward
genetic screen, 1 ? 106168FARN-Luc cells were orthotopically
transplanted into the mammary fat pads of 10-week-old BALB/cJ
mice or injected intravenously by tail vein. Mice bearing luciferase
Xenogen IVIS system (Xenogen Corporation, Hopkinton, MA).
Mice injected by tail vein were imaged 4–5 weeks after transplan-
genetic screen, retroviruses containing a cDNA library were gen-
viruses containing the library at 1 multiplicity of infection.
168FARN-Luc cells (1 ? 106) containing the library were trans-
planted into the mammary fat pad of each mouse. Lung metastasis
nodules were isolated 6–7 weeks after transplantation. Genomic
DNA of metastatic cells was isolated with the Genomic DNA
Purification kit according to supplier’s instruction (Qiagen, Valen-
cia, CA). PCRs were performed with the Expand High Fidelity kit
(Roche Diagnostics, Indianapolis, IN) with primers as described
(23). PCR products were cloned with a TA cloning kit (Invitrogen)
cells were infected with retroviruses containing the full-length
mouse ERp5 behind the LTR promoter; 1 ? 106cells were
transplanted into the mammary fat pads of mice and imaged 6–8
weeks after transplantation.
We thank Dr. Fred Miller for 168FARN cells and Dr. George Daley for
the cDNA library. Q.H. is supported by the Breast Cancer Alliance, the
Pardee Foundation, and the Commonwealth Universal Research En-
hancement Program (Pennsylvania Department of Health). We also
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