© The American Society of Gene & Cell Therapy
original article
Multiple Functions of the 37/67-kd Laminin
Receptor Make It a Suitable Target for Novel
Cancer Gene Therapy
Jonathan Scheiman1, Jen-Chieh Tseng1, Yun Zheng1 and Daniel Meruelo1
NYU Cancer Institute and the NYU Gene Therapy Center, NYU School of Medicine, New York, New York, USA
1
The 37/67-kd laminin receptor, LAMR, is a ­multifunctional
protein that associates with the 40S ribosomal sub-
unit and also localizes to the cell membrane to inter-
act with the extracellular matrix. LAMR is overexpressed
in many types of cancer, playing important roles in
tumor-cell migration and invasion. Here, we show that
LAMR is also vital for tumor-cell proliferation, survival,
and protein translation. Small-interfering RNA (siRNA)–
mediated reduction in expression of LAMR leads to G1
phase cell-cycle arrest in vitro by altering cyclins A2/B1,
cyclin-dependent kinases (CDKs) 1/2, Survivin, and p21
expression levels. In vivo, reduction in LAMR expression
results in inhibition of HT1080 cells to develop tumors.
We also found that LAMR’s ribosomal functions are criti-
cal for translation as reduction in LAMR expression leads
to a dramatic decrease in newly synthesized proteins.
Further, cells with lower expression of LAMR have fewer
40S subunits and 80S monosomes, causing an increase
in free 60S ribosomal subunits. These results indicate
that LAMR is able to regulate tumor development in
many ways; further enhancing its potential as a target
for gene therapy. To test this, we developed a novel
Sindbis/Lenti pseudotype vector carrying short-hairpin
RNA (shRNA) designed against lamr. This pseudotype
vector effectively reduces LAMR expression and specifi-
cally targets tumors in vivo. Treatment of tumor-bearing
severe combine immunodeficient (SCID) mice with this
pseudotype vector significantly inhibits tumor growth.
Thus, we show that LAMR can be used as a target in
novel therapy for tumor reduction and elimination.
Received 11 October 2009; accepted 28 July 2009; published online
1 September 2009. doi:10.1038/mt.2009.199
Introduction
The 37/67-kd laminin receptor (LAMR) plays a role in many path-
ological processes. It serves as a cell-surface receptor for prions,1
cytotoxic necrotizing factor-1 expressing Escherichia coli K1 (ref. 2)
and numerous viruses, including Sindbis,3 dengue,4 Venezuelan
equine encephalitis,5 and adeno-associated virus subtypes 2, 3,
8, and 9 (ref. 6). LAMR is also over expressed in many types of
Correspondence: Daniel Meruelo, NYU Cancer Institute and the NYU Gene Therapy Center, NYU School of Medicine, 550 First Avenue, New York,
New York 10016, USA. E-mail: merued01@med.nyu.edu
Molecular Therapy vol. 18 no. 1, 63–74 jan. 2010
cancer7–12 where as an extracellular matrix molecule, it is important
for tumor-cell migration,13 invasion, and angiogenesis.14 Further,
LAMR can remodel laminin-1 to alter tumor-cell gene expression
and increase tumor aggressiveness,15 as well as alter intracellular
signaling pathways.16 LAMR is therefore considered a useful prog-
nostic marker for determining the severity of tumors.17
The 67-kd LAMR was first discovered by three independent
laboratories in 1983 (refs. 18–20) through its ability to bind to and
be isolated by laminin sepharose. Its gene, however, was found to
encode a protein of only 37 kd. The discrepancy between these two
molecular weights was later resolved by showing that the 37-kd
gene product serves as a monomeric precursor to a 67-kd ­dimer.21
The exact composition of the 67-kd dimer and the process by
which it is formed remains obscure as evidence supports both a
homo22 and a heterodimer.23,24 LAMR was shown to be acylated by
three fatty acids—palmitate, stearate, and oleate22—and fatty acid
synthesis is required for 67-kd LAMR formation.23 Beyond this not
much is known about what regulates the dimerization process.
The 37-kd LAMR monomer is not without its own intrigue.
It is a highly conserved ribosomal protein that acquired its extra-
cellular matrix functions during evolution.25 The 37-kd LAMR,
also known as p40, is ubiquitously expressed in many organisms
as a 40S ribosome–associated protein. The mammalian sequence
has homologues in many different organisms including bacteria,
yeast, plant (reviewed in ref. 25). The 37-kd LAMR/p40 has been
shown to be polysome associated in both yeast26 and plant.27 The
ribosomal functions of 37-kd LAMR are essential for cell viability
in yeast; 37-kd LAMR/p40 is required for processing 20S to 18S
ribosomal RNA and thus for maturation of the 40S ribosomal sub-
unit and 80S monosome assembly.28 The 37-kd LAMR/p40 is also
required for HeLa29 and Hep3b30 cell viability, although in HeLa
cells loss of 37-kd LAMR/p40 reportedly did not inhibit protein
translation and therefore was thought not to be the cause of cell
death. LAMR has also been shown to localize to the nucleus and
interact with histones H2A, H2B, and H4 (ref. 31). Thus nuclear
localization/functions of LAMR may play a critical role in cell
viability. However, the fact that LAMR is polysome associated in
mammalian cells32 suggests that it retains some ribosomal func-
tion. The importance of 37-kd LAMR as a ribosomal protein and
how it maintains cell viability in mammalian cells still needs to be
thoroughly examined and is something we wanted to investigate.
63

LAMR as a Target for Novel Cancer Gene Therapy
Considering the large number of cellular processes that
LAMR governs and the overwhelming evidence for its involve-
ment in pathology, further understanding of its functionality
is important for developing it as a target for new therapeutic
strategies. LAMR is already a target for prion disease therapy.33
Also, many studies have focused on the extracellular functions
of LAMR for cancer therapeutic purposes. For instance, using
various anti-LAMR agents impede tumor-cell invasion in vitro34
and antibodies that specifically bind LAMR can inhibit tumor
metastasis in vivo.35 Currently, it is not known what role the 37-kd
monomer plays in tumor development. Targeting intracellular
and ribosomal functions of 37-kd LAMR might be as effective as
inhibiting extracellular functions although has been explored to a
lesser extent. Therefore, we seek to examine how the 37-kd LAMR
monomer may effect tumor development using small-interfering
RNA (siRNA) to silence lamr expression and to assess whether we
can improve upon current strategies that target LAMR solely as
an extracellular molecule. Silencing total lamr expression might
inhibit multiple LAMR functions and therefore more effectively
address questions about cell viability as well as be more efficient
in treating tumors in vivo. In this study, we focus exclusively on
the expression of the 37-kd monomer, and therefore refer to the
monomer whenever we mention LAMR.
Targeting genes for cancer gene therapy in vivo, as we seek
to do with LAMR, has hitherto been greatly limited by lack of an
efficient delivery system for tumor targeting. A vector system is
needed to achieve specific and efficient tumor targeting in living
animals. Several vectors based on lentivirus (e.g., human immu-
nodeficiency virus) have been developed to deliver foreign DNA
into cells without significant safety concerns. The core of lentivi-
rus can accommodate a wide range of glycoproteins from other
viruses, providing a relatively flexible choice of viral glycoprotein
for specific targeting. Mentioned earlier, LAMR is a cellular recep-
tor for Sindbis virus. We have previously developed Sindbis viral
vectors as therapeutic agents to specifically target and kill tumor
cells and have also shown that Sindbis’ tumor-targeting ability
is mediated in part by LAMR.36 Thus, not only is LAMR itself a
potential focus for cancer therapy, it also is a means of targeting
tumors with Sindbis viral vectors. Here, we explore the use of a
Sindbis/Lenti pseudotype vector, carrying a short-hairpin RNA
(shRNA) cassette, to target tumors cells in vivo and effectively
reduce LAMR expression for cancer gene therapy.
Results
Knocking down LAMR expression inhibits
cell proliferation
To determine the role that LAMR plays in tumor-associated func-
tions, we transiently knocked down its expression in the HT1080
human fibrosarcoma cell line by transfection with a predesigned
siRNA pool targeting human LAMR (siLAMR). This pool consists
of four individual oligonucleotides that target different regions
of the human LAMR sequence (Supplementary Figure S1).
Fluorescently labeled nontargeting control siRNA (siGLO) was
used as a control. Fluorescent activated cell sorting analysis shows
that ~97% of HT1080 cells are transfected with siGLO (Figure 1a),
indicating a high level of siRNA transfection efficiency. We ana-
lyzed the efficiency of LAMR knock down via siLAMR by both
64
© The American Society of Gene & Cell Therapy
quantitative real-time PCR and western blot analysis (Figure 1b
left and right, respectively). In our system, we are able to achieve
>90% knock down of LAMR expression at both the mRNA and
protein levels.
We observed that siLAMR-transfected cells grow slower than
control-transfected cells (Figure 1c, left). Cell proliferation was
therefore assessed. Equal numbers of nontransfected, siLAMR-,
and siGLO-transfected cells were seeded 2 days after transfection
and allowed to grow for 3 days after seeding. As Figure 1c shows
(right), there was a twofold reduction in siLAMR-transfected
cell number compared to control cells 1 day after seeding cells,
­followed by a 5- and 6.5-fold reduction 2 and 3 days after seeding,
respectively. Thus, cells with reduced expression of LAMR have a
lower proliferation rate. This phenomenon was also observed with
293, HeLa, and HepG2 cells (Figure 1d) suggesting that LAMR
plays an important role in cellular growth rate.
Reduction in LAMR expression results in G1 phase
cell-cycle arrest
Cell-cycle profiles of nontransfected, siLAMR-, and siGLO-trans-
fected cells were compared 3–6 days after transfection by stain-
ing with propidium iodide and analyzing by fluorescent activated
cell sorting. Profiles are summarized in Figure 2a. At 4 days after
transfection, ~40% of both nontransfected and siGLO cells were in
G1 phase compared to about 55% for the siLAMR-transfected cells
(Figure 2a, left panel) and ~40% of the control cells are in S phase
compared to about 25% of siLAMR-transfected cells (Figure 2a,
right panel). The disparity becomes even greater at 5 days after
transfection (Figure 2b) when 30% of nontransfected and 28% of
siGLO-transfected cells were in the G1 phase compared to 65%
of siLAMR-transfected cells. Furthermore, 55% of nontransfected
and 49% of siGLO-transfected cells were in S phase compared to
only 16% of the siLAMR-transfected cells. These results indicate
that cells with reduced levels of LAMR undergo cell-cycle arrest
in the G1 phase. The arrest is only transient, however, as the cell-
cycle profiles of siLAMR-transfected cells are similar to those of
the control cells 6 days after transfection as shown in Figure 2a.
LAMR expression affects expression levels of cell
cycle–related genes
Because siLAMR-transfected cells undergo cell-cycle arrest,
we investigated whether any cell cycle–related gene ­expression
is altered. Using the cell cycle RT2Profiler PCR array from
SuperArray (Frederick, MD), the expression levels of 84 different
genes were compared between siLAMR- and siGLO-transfected
cells. Several genes were found whose expression was altered in
siLAMR-transfected cells in a manner consistent with G1 arrest
(data not shown). These genes, validated by quantitative real-time
PCR at 3 and 4 days after transfection (Figure 3a left and right,
respectively), included cyclins A/B, cyclin-dependent kinases
(CDKs) 1/2, E2F1, and p21.
At the mRNA level, p21, a cyclin-dependent kinase inhibitor
and tumor suppressor gene that inhibits cell-cycle progression
at the G1/S check point, was upregulated in siLAMR-transfected
cells 15-fold 3 days after transfection and eightfold 4 days after
transfection (Figure 3a left and right, respectively). In contrast,
cyclin A2, expressed in S/G2, and cyclin B1, expressed in G2/M
www.moleculartherapy.org vol. 18 no. 1 jan. 2010
© The American Society of Gene & Cell Therapy
LAMR as a Target for Novel Cancer Gene Therapy

a 100
siGLO
80

% of Max
60
97.2
40 Non-
transfected siLAMR siGLO

20 LAMR

0
100 101 102 10
3
104 β-Actin
FL2-H

b LAMR mRNA expression LAMR protein expression

Nontransfected Nontransfected
2.5
1.5 siLAMR
siLAMR
siGLO 2.0 siGLO

1.0

Fold change
Fold change

1.5

1.0
0.5
0.5

*** ***
0.0 0.0

c Nontransfected siGLO siLAMR

HT1080
7,500,000
Day 1 Nontransfected
Relative light units

siLAMR
5,000,000 siGLO

Day 2 2,500,000

0
1 2 3
Day 3 Time (days)

d 293 HeLa HepG2

Nontransfected 30,000,000 30,000,000
100,000,000
siLAMR
Relative light units

siGLO
75,000,000 20,000,000 20,000,000

50,000,000
10,000,000 10,000,000
25,000,000

0 0 0
1 2 3 4 5 1 2 3 4 1 2 3 4 5
Time (days)

Figure 1 Reduction in laminin receptor (LAMR) expression inhibits cell proliferation. (a) HT1080 cells transfected with the fluorescently labeled
nontargeting small-interfering RNA (siRNA) control, siGLO, were analyzed with a FACSCaliber machine and gated according to nontransfected cells
(dotted line). Approximately 97% of siGLO-transfected cells (solid line) were positive for siGLO, as determined by Flowjo 8.2 software (Tree Star).
Fluorescent activated cell sorting analysis was performed 1 day after transfection. (b) HT1080 cells transfected with an siRNA pool targeting LAMR
(siLAMR) were harvested for RNA 3 days after transfection and protein extraction 4 days after transfection. Quantitative real-time PCR was used to
check LAMR mRNA expression (left) and western blot analysis was used to check LAMR protein levels (top right), using glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and β-actin as internal controls, respectively. Western blot results were quantified and graphed (bottom right). Statistical
analysis was performed using a standard Student’s t-test to generate P values. All P values are two-tailed (***P < 0.0001). (c) Images of nontransfected,
siLAMR-, and siGLO-transfected cells were taken 1–3 days after seeding for proliferation assay (3–5 days after transfection, respectively) (left). Cell pro-
liferation was measured for HT1080 nontransfected, siLAMR-, and siGLO-transfected cells at the same time points (right). (d) Similar cell ­proliferation
assays were performed for 293, HeLa, and HepG2 cell lines.

phases were downregulated 90% at both time points. CDKs1 and when comparing siLAMR-transfected cells to siGLO-transfected
2, which promote cell-cycle progression, are both reduced ~60% cells. At the mRNA level, there was generally no significant dif-
3 days after transfection (Figure 3a, left). In addition, the E2F1 ference between nontransfected and siGLO-transfected cells
transcription factor, expressed during S phase, was downregu- in the genes analyzed. In the few exceptions when there was,
lated ~60% 3 days after transfection (Figure 3a, left). The statis- it only enhanced the disparity between siLAMR- and siGLO-
tical significance of each gene was evaluated to generate P values ­transfected cells.

Molecular Therapy vol. 18 no. 1 jan. 2010 65

 © The American Society of Gene & Cell Therapy
LAMR as a Target for Novel Cancer Gene Therapy

a Percentage of HT1080 cells in Percentage of HT1080 cells in

G1 phase S phase
75 75
Nontransfected
siLAMR
% 50 50 siGLO

25 25

0 0
3 4 5 6 3 4 5 6
Time (days after transfection)

b Nontransfected siLAMR siGLO

800 800 800

600 600 600

Cell number

G1 G1 G1
400 30% 400 65% 400 28%
S 55% S 16% S 49%
200 200 200

0 0 0
0 30 60 90 120 150 0 30 60 90 120 150 30 60 90 120 150
Channels

Figure 2 siLAMR-transfected cells undergo cell-cycle arrest in the G1 phase. (a) Summary of cell-cycle profiles for HT1080 siRNA-transfected
cells. Cells were stained with propidium iodide and analyzed by fluorescent activated cell sorting. Percentage of cells in G1 phase (left) and S
phase (right) are shown 3–6 days after transfection. (b) Cell-cycle profiles 5 days after transfection with siRNA. Percentage of cells in G1 and S phase
are indicated (arrows). siGLO, fluorescently labeled nontargeting siRNA control; siLAMR, siRNA pool targeting human laminin receptor; siRNA, small
interferin RNA.

Protein levels of these gene products were measured 4 days
after transfection. As Figure 3b (left) shows, knock down of
LAMR resulted in a dramatic decrease in cyclins A/B, and
CDKs 1/2 at the protein level. On the other hand, p21, barely
expressed in control cells, was upregulated in siLAMR-trans-
fected cells. β-Actin was used as a loading control and shows
that equal amounts of protein were loaded. Western blots are
representative of at least three transfections. Protein ­expression
levels were quantified and statistically analyzed comparing siL-
AMR- and siGLO-transfected cells (Figure 3b, right). These
results indicate that reduction in LAMR expression alters the
expression of genes and proteins that are important for cell-
cycle progression.
Another gene shown to be downregulated in the Cell-cycle
SuperArray and validated through real-time PCR was Survivin.
An antiapoptotic protein overexpressed in many types of cancer,
Survivin was reduced 90% both at the mRNA level and protein
level in siLAMR-transfected cells (Figure 3a,b). At the protein
level, there is also a significant reduction of Survivin in siGLO-
transfected cells compared to nontransfected cells, which may be
due to the transfection procedure. However, the fact that Survivin
is reduced at the mRNA level only in siLAMR-transfected cells
and almost completely absent in siLAMR-transfected cells at the
protein level suggests that reduction of Survivin expression is a
specific effect of LAMR knock down. In addition, it also suggests
that tumor cells with reduced levels of LAMR may also be more
susceptible to cell death.
LAMR expression regulates translation
Because LAMR is a ribosomal protein, we wanted to determine
whether it plays a role in translation in HT1080 cells. Novo pro-
tein synthesis by nontransfected, siLAMR- and siGLO-trans-
fected cells was measured 2–4 and 7 days after transfection by
pulsing cells with 35S-methionine for 2 hours and chasing for an
additional 90 minutes. Equal amounts of protein were loaded for
sodium dodecyl sulfate–polyacrylamide gel electrophoresis and
used to detect newly synthesized proteins. As Figure 4a shows,
translation was markedly inhibited in siLAMR-transfected cells
at all time points, indicating that LAMR expression is critical
for this process. In addition, treating siLAMR-transfected cells
with trypsin and reseeding them the day before labeling had no
­additional effect on translation inhibition, indicating that this
process is not regulated by extracellular functions of LAMR
important for cell adhesion.
LAMR was previously shown to be required for 40S ribo-
somal subunit maturation and 80S monosome assembly in
yeast.28 To analyze ribosomal integrity in siLAMR- and siG-
LO-transfected cells, sucrose-gradient ultracentrifugation was
used. Equal numbers of cells were lysed and separated through
10–50% linear sucrose gradients 2 and 3 days after transfection.
A total of 24 fractions of equal volume were collected from the
top of each gradient and their optical density was measured
at A260 nm to generate ribosomal profiles (Figure 4b). Two days
after transfection, there was a modest reduction in the 80S
monosome peak in siLAMR-transfected cells (Figure 4b, left).

66 www.moleculartherapy.org vol. 18 no. 1 jan. 2010

© The American Society of Gene & Cell Therapy
LAMR as a Target for Novel Cancer Gene Therapy

a HT1080 siLAMR HT1080 siLAMR

Cell cycle gene expression Cell cycle gene expression
(3 days) (4 days)
Nontransfected
15.0 9
siLAMR
12.5 siGLO 7

10.0 5
Fold change

1 1

0 0
R

A2

B1

K1

K2

21

A2

B1

K2

F1

in

21
2F

vi
AM

iv
*p

*p
E2
D

D
vi
lin

lin

lin

lin

rv
LA
*E
*C

*C
ur
*L

Su
**
yc

yc

yc

yc
**
*S
**
*C

*C

**
**

**
Gene

b Nontransfected siLAMR siGLO

LAMR HT1080 siLAMR
(37 kd) Cell cycle protein expression

Cyclin A2 Nontransfected
5
(50 kd)
siLAMR
4
Cyclin B1 siGLO
(50 kd) 3

CDK1 2
Fold change

(34 kd) 1.5

CDK2
(34 kd) 1.0

Survivin
0.5
(15 kd)

p21 0.0
(21 kd)
R

A2

B1

K1

K2

in

21
M

iv

*p
D

D
lin

lin

rv
LA

*C

**
Su
yc

yc

**
**

**

β-Actin
*C

**
**
**

(42 kd) Protein

Figure 3 siLAMR alters the expression of cell cycle–related genes and proteins. (a) Quantitative real-time PCR validation of cell cycle–related
genes found to be altered in HT1080 siLAMR–transfected cells 3 days (left) and 4 days (right) after transfection. Glyceraldehyde-3-phosphate dehy-
drogenase was used as an internal control. Results are indicative of at least two separate transfections. (b) Western blot analysis of cell cycle–related
proteins 4 days after transfection. Protein (20 μg) was loaded. β-Actin was used as a control. Western blots are indicative of three separate trans-
fections (left). Observed molecular weights of each protein are indicated in parentheses. Protein expression levels were quantified and statistically
­analyzed (right) (*P < 0.05. **P < 0.001. ***P < 0.0001). CDK, cyclin-dependent kinase; siGLO, fluorescently labeled nontargeting siRNA control;
siLAMR, siRNA pool targeting human LAMR; siRNA, small interferin RNA.

Three days after transfection, the 80S monosome peak was
dramatically reduced in siLAMR-transfected cells, which was
accompanied by a reduction in the 40S peak and an increase in
the 60S peak (Figure 4b, right).
Fractions 1–12 from the 3-day time point were further ana-
lyzed by western blot, probing for LAMR, 40S ribosomal pro-
tein S6, and 60S ribosomal protein L7a. As Figure 4c shows,
­reduction in LAMR resulted in a shift of L7a from fractions 9
and 10, which corresponds with the 80S monosome peak in
the siGLO-transfected ribosomal profile, to fractions 7 and 8,
­corresponding to the 60S subunit fractions. S6 was also reduced
in fractions 9 and 10. These blots therefore confirmed the ribo-
somal profiles, and show that LAMR is crucial for 40S matu-
ration and 80S ­monosome assembly in HT1080 cells. Without
LAMR, there is a reduction in 40S subunits, which results in
an increase of unassociated 60S ribosomal subunits, less 80S
monosomes, and a dramatic ­reduction in translation as men-
tioned above.
LAMR is critical for tumor growth in vivo
To further examine the effects of knocking down LAMR expres-
sion, we assessed the ability of cells with reduced levels of LAMR
to grow tumors in vivo. An HT1080 cell line was generated
that stably expresses the firefly luciferase gene (HT1080 FLUC)
­permitting it to be visualized and quantified in vivo with use of
an in vivo imaging system (IVIS). A volume of 1 × 106 nontrans-
fected, siLAMR-, and siGLO-transfected HT1080 FLUC cells
were injected subcutaneously into mice 2 days after transfection
(five mice were injected separately for each group). In addition,
cells were reseeded in tissue culture and harvested for RNA 4
days after transfection. siLAMR-transfected cells had over an 80%
reduction of LAMR mRNA compared to siGLO-transfected cells
(Figure 5a).
Mice were imaged 3, 10, 17, and 24 days after injection (5, 12,
19, and 26 days after transfection, respectively) to monitor tumor
development of HT1080 FLUC fibrosarcoma cells (Figure 5b,
left). Tumor-cell fluorescence, which stayed localized to the sight

Molecular Therapy vol. 18 no. 1 jan. 2010 67

 © The American Society of Gene & Cell Therapy
LAMR as a Target for Novel Cancer Gene Therapy

a 2 days 3 days

35
S-methionine

LAMR

β-Actin
Non- siLAMR siGLO Non- siLAMR siLAMR siGLO
transfected transfected Trypsin

4 days 7 days

35
S-methionine

LAMR
β-Actin
Non- siLAMR siLAMR siGLO Non- siLAMR siLAMR siGLO
transfected Trypsin transfected Trypsin

b 1.4 2 days
1.2
3 days

1.2 1 siGLO
1 siLAMR
0.8
0.8 40S 60S 80S 40S 60S 80S
A260

0.6
0.6
0.4
0.4

0.2 0.2

0 0
0 5 10 15 20 25 0 5 10 15 20 25
Fraction

c siGLO siLAMR
LAMR

L7a

S6
4 5 6 7 8 9 10 4 5 6 7 8 9 10
Fraction

Figure 4 Laminin receptor (LAMR) expression regulates translation. (a) Nontransfected, siLAMR-, and siGLO-transfected cells were metabolically
labeled with 35S-methionine, 2–4, and 7 days after transfection. Labeling of cells was performed in triplicate. Protein (20 μg) was loaded for sodium
dodecyl sulfate–polyacrylamide gel electrophoresis to detect newly synthesized proteins. siLAMR-transfected cells were either allowed to remain
attached to dishes or treated with trypsin the day before labeling and then reseeded (siLAMR trypsin). Western blot analysis was used to check for
LAMR and β-actin expression. (b) An equal number of siLAMR- and siGLO-transfected cells were collected and lysed 2 and 3 days after transfection.
Cell lysates were separated on a 10–50% linear sucrose gradient and collected in 24 fractions of equal volume. The optical density of each fraction
was measured at A260 nm to generate ribosomal profiles. Solid lines represent siLAMR-transfected profiles, dashed lines represent siGLO-transfected
profiles. Arrows indicate 40S, 60S, and 80S peaks. (c) Thirty-two microliters of fractions 1–12 from the siLAMR and siGLO ribosomal gradients, 3 days
after transfection, were loaded for western blot analysis. Membranes were blotted for LAMR, L7a, and S6. siGLO, fluorescently labeled nontargeting
siRNA control; siLAMR, siRNA pool targeting human LAMR; siRNA, small interferin RNA.

of injection, was then quantified (Figure 5b, right). At 3 days after
injection, control cells had an average fluorescence about two-
fold higher than siLAMR-transfected cells, which increased to
1,000-fold after 10 days, consistent with the higher proliferation
rate of control versus siLAMR-transfected cells seen in vitro. At
later time points, siLAMR-transfected tumors show a decrease in
luciferase signal intensity and by 17 days the siLAMR-transfected
tumor cells can no longer be detected (Figure 5b). This indicates
that siLAMR-transfected HT1080 cells have an impaired ability to
grow in vivo. In contrast to the other experimental groups, animals

68 www.moleculartherapy.org vol. 18 no. 1 jan. 2010

© The American Society of Gene & Cell Therapy
LAMR as a Target for Novel Cancer Gene Therapy

a HT1080 FLUC
LAMR mRNA expression
1.0 Nontransfected
siLAMR
siGLO

Fold change
0.5

0.0 ***

b Day 3 10 17 24
Nontransfected

60,000 HT1080 tumor growth curves

50,000 5.0 × 107

40,000 4.0 × 107

Photon counts
30,000 3.0 × 107
siLAMR

20,000 Nontransfected (n = 5)
2.0 × 107
10,000 siLAMR (n = 5)
7
1.0 × 10
siGLO (n = 5)
Counts
0
Color Bar 0 10 20 30 40 50 60
Min = 600 Days after inoculation
Max = 60,000
siGLO

Figure 5 LAMR is critical for HT1080 tumor-cell growth and survival in vivo. (a) HT 1080 FLUC nontransfected, siLAMR-, and siGLO-transfected
cells were harvested for RNA extraction 4 days after transfection. Quantitative real-time PCR was performed to determine LAMR mRNA expression
levels. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. (***P < 0.0001). (b) Severe combine immunodeficient mice
were injected subcutaneously with HT1080 FLUC nontransfected, siLAMR-, and siGLO-transfected cells and allowed to grow tumors. Luciferase signal
intensity was measured 3, 10, 17, and 24 days after injection with use of in vivo imaging system (left) and quantified (right). Luciferase signal intensity
was also quantified 55 days after injection for siLAMR-transfected cells. siGLO, fluorescently labeled nontargeting siRNA control; siLAMR, siRNA pool
targeting human LAMR; siRNA, small interferin RNA.

receiving siLAMR-transfected tumor cells remained healthy for
the duration of the experiment and after tumor cells regressed
(by days 17 or so) did not display luciferase signal by IVIS or any
symptoms associated with recurrence of tumor growth. This dem-
onstrates that LAMR expression plays a major role in the growth
and survival of HT1080 tumors. These results also show, targeting
and inhibiting LAMR expression in vivo is may be a promising
strategy for treating cancer.
Targeting LAMR with Sindbis/Lenti pseudotype
vectors inhibits tumor growth in vivo
As a proof of principle for altering LAMR expression to treat
tumors, we explored the possibility of reducing lamr expression in
tumor cells in vivo in a targeted manner. We utilized our previous
experience with Sindbis viral vectors to develop a novel pseudo-
type Sindbis/Lenti vector combining the core proteins of lentivi-
rus and the structural proteins of Sindbis virus (Figure 6a). To
test tumor-targeting capabilities, we used a previously developed
advanced ovarian cancer animal model.36 Severe combine immu-
nodeficient (SCID) mice were injected intraperitoneally (i.p.) with
the ES-2 ovarian carcinoma cell and allowed to develop tumors
in the peritoneal cavity. SCID mice with ES-2-derived tumors
and mice without tumors were then treated with a pseudotype
Sindbis/Lenti vector that expresses the firefly luciferase gene. As
Figure 6b (left) shows, only mice injected with ES-2 cells and have
tumors show a luciferase signal during mouse whole-body imag-
ing. Further, organs from mice with ES-2 tumors were harvested
from the peritoneal cavity and show that ES-2 tumors and pseudo-
type Sindbis/Lenti vector localize primarily to the intestine. These
findings are consistent with our previous results using this model
with Sindbis viral vectors. Our pseudotype vector therefore has
the same tumor-targeting capabilities as Sindbis viral vectors and
can be used for cancer gene therapeutics.
We next generated two pseudotype vectors that carry an
shRNA cassette, one specific for lamr and another, as a control,
specific for LacZ. In vitro transduction of ES-2 ovarian carcinoma

Molecular Therapy vol. 18 no. 1 jan. 2010 69

 © The American Society of Gene & Cell Therapy
LAMR as a Target for Novel Cancer Gene Therapy

cells with the pseudotype vector specific for lamr reduces LAMR Sindbis/Lenti pseudotype vector to achieve this, SCID mice with
protein expression by ~50% compared to the control vector that tumors derived from ES-2 cells expressing the firefly luciferase
targets LacZ (Figure 6c). Western blots are indicative of at least gene were treated with either the control pseudotype vector or
three independent transductions. To test both the efficacy of reduc- pseudotype vector that reduces LAMR expression. Tumor growth
ing LAMR expression for gene therapy as well as the ability of our was measured by luciferase signal intensity 1 day after injection of

a pLP/E321 PCMV β-Globin intron Sindbis E321 β-Globin pA

b Tumor free Tumor bearing

Sindbis/Lenti
packaging pLP1 PCMV β-Globin intron Gag/Pol RRE β-Globin pA
plasmids

pLP2 PRSV Rev β-Globin pA

PRSV/5’LTR ψ RRE PSV40 EM7 Blasti ∆U3/3’LTR SV40 pA

Expression cassettes
pLenti6/Fluc PUBC Firefly luciferase gene

Lentiviral
expression pLenti6/shLacZ PU6 LacZ shRNA Pol III term
plasmids

pLenti6/shLAMR PU6 LAMR shRNA Pol III term

c LacZ shLAMR
LAMR

β-Actin

HT1080 shLAMR
LAMR protein expression

1.0 LacZ
shLAMR
Fold change

**
0.5

0.0
d Sindbis/Lenti-
shLacZ shLAMR
Day 4 growth percentage
300
Day 1

200
Growth %

60,000 Luciferase whole-body counts

75,000,000

50,000
100 * shLacZ

shLAMR
40,000
50,000,000
Photon counts

0
shLacZ shLAMR
30,000 Treatment
Day 4

20,000 Day 11 growth percentage

3,000 25,000,000

10,000

2,000
Growth %

Counts 0
0 2 4 6 8 10 12
Color Bar
Min = 1,000
* Day
1,000
Max = 60,000
Day 11

0
shLacZ shLAMR
Treatment

70 www.moleculartherapy.org vol. 18 no. 1 jan. 2010

© The American Society of Gene & Cell Therapy
LAMR as a Target for Novel Cancer Gene Therapy

ES-2 tumor cells and before pseudotype vector treatment (day 1)
as well as 3 and 10 days after treatment (day 4 and day 11 after ES-2
tumor-cell injection, respectively) (Figure 6d, left). Luciferase
­signal intensity after treatment was compared to signal intensity
pretreatment to determine tumor growth percentage for day 4 and
day 11 (Figure 6d, middle, top, and bottom, respectively). Mice
receiving treatment with pseudotype vector that targets lamr show
~60 and 50% reduced tumor growth compared to control pseudo-
type vector on days 4 and 11, respectively. Luciferase whole-body
counts from all days imaged were used to generate tumor growth
curves (Figure 6d, right). The finding that Sindbis/Lenti pseudo-
type vector that reduces LAMR expression also reduces tumor-
cell growth is similar to our previously described results with
the HT1080 siLAMR–transfected tumor model. Thus, utilizing a
novel vector and approach to cancer gene therapy, we demonstrate
that altering LAMR expression can be used for cancer therapy.
Discussion
Extensive efforts have been made to determine the role of LAMR
in tumor development. Many studies, such as Zuber et al., have
focused on extracellular functions including migration and inva-
sion. Experiments performed by our lab have similarly observed
that LAMR is important for both of these processes (data not
shown). However, our finding that siLAMR-transfected cells have
a limited capacity to proliferate prompted further investigation of
LAMR’s potential role in tumor-cell growth. LAMR expression is
essential for cell growth and viability in yeast, and was shown to be
critical for ribosome maturation and assembly. Our results have
found concordance with the yeast studies and clearly show LAMR
functions in mammalian cells as a ribosomal protein crucial for
protein translation.
Protein translation is important for G1 to S phase cell-cycle
progression. The fact that a reduction in LAMR expression leads
to translation inhibition and G1 cell-cycle arrest indicates LAMR’s
ribosomal functions can regulate cell proliferation. However, we do
observe that siLAMR-transfected cells start to recover from growth
arrest at a time in which translation is still inhibited, suggesting
that perhaps cell proliferation may be regulated by a nonribosomal
function of LAMR. It should be noted that the level of translation
inhibition appears to be less severe 7 days after ­siLAMR transfec-
tion than at earlier time points when cells are growth arrested. It is
possible that the first proteins to be ­translated as cells recover are
those important for cell-cycle progression. In this case, cells would
start to proliferate even as other protein expression is still inhib-
ited. Conversely, we observe that protein expression is differentially
regulated in order to induce growth arrest, as can seen by the fact
that p21 protein levels actually increase while translation as a
whole is inhibited. It may be that LAMR expression is important
for translation of a majority of proteins but not all. Further, LAMR
expression may promote the translation of proteins important for
cell-cycle progression, while a reduction in LAMR expression may
lead to translation of proteins that induce growth arrest, such as
p21. Interestingly, it was shown that LAMR/p40 has a higher affin-
ity for polysomes in plant tissues during times of active growth.37
Therefore, LAMR may only be essential for translation of proteins
that are required for proliferation.
It is also important to keep in mind that we are working with
a transient system, and therefore see slight variations with each
transfection. Although we find that siLAMR-transfected cells are
always growth arrested, the duration of growth arrest is variable.
Likewise, we notice that there are times when siLAMR transfec-
tion has a dramatic effect on cell morphology in vitro (data not
shown), but this is not always the case. Because of this, the kinetics
and specifics of our transient siLAMR system are currently under
further investigation.
Despite these questions in vitro, the effect of siLAMR in vivo
is much more profound, indicating that the ribosomal functions
of LAMR may be more critical for tumor-cell survival in a more
physiological environment. The ability of LAMR to regulate transla-
tion, proliferation, and survival as a ribosomal protein may there-
fore be as important, if not more, than its extracellular functions
in tumor development. This has been suggested by several studies.
For example, the levels of LAMR upregulation in cervical tumors
resulting from papiloma virus infection ­correlates with increased
cell proliferation but not invasion.38 Also, while LAMR serves as
the cellular receptor for the green tea polyphenol (−)-epigallocat-
echin-3-gallate,39 another ribosomal protein, eukaryotic translation
elongation factor 1A, is also required for (−)-epigallocatechin-3-
­gallate-induced tumor cell death.40 In addition, (−)-epigallocate-
chin-3-gallate specifically targets multiple myeloma cell in vivo via
LAMR to induce apoptosis and growth arrest in the G1 phase of the
cell cycle.41 Therefore, the ribosomal functions of LAMR may also be
important for (−)-epigallocatechin-3-gallate-induced effects. These
and our observations add LAMR to a growing list of ribosomal pro-
teins, in which overexpression promotes cell growth and subsequent
loss of LAMR expression (or inhibition) results in cell death.42
In this work, our goals were to determine what role LAMR plays
in translation and tumor viability/development as well as to test its
therapeutic potential as a target for cancer gene therapy. Recently, it
was shown that LAMR expression can be reduced in vivo through

Figure 6 Targeting laminin receptor (LAMR) with Sindbis/Lenti pseudotype vectors inhibits ES-2 tumor growth in vivo. (a) Schematic diagram
of pseudotype vector construct. Notably, vesicular stomatitis virus-G structural proteins have been replaced by Sindbis E1, E2, and E3 structural
proteins. (b) Severe combine immunodeficient (SCID) mice without tumors (tumor free) and SCID mice with peritoneal tumors derived from ES-2
ovarian carcinoma cells (tumor bearing) were treated with a pseudotype Sindbis/Lenti-FLUC vector. In vivo imaging system (IVIS) was used to detect
luciferase expression (pseudotype vector infection) by whole-body imaging (left). Peritoneal organs were extracted from SCID mice with ES-2 tumors
treated with pseudotype Sindbis/Lenti-FLUC and imaged with IVIS to show tumor infection localization (K, kidney; L, liver; S, spleen; I, intestine)
(right). (c) ES-2 ovarian carcinoma cells were transduced in vitro with a Sindbis/Lenti pseudotype vector that carries an shLAMR cassette against lamr
or LacZ. Western blot analysis was used to check expression levels of LAMR and β-actin. Western blots are indicative of at least three separate infections
(top). Protein expression levels were quantified and statistically analyzed (bottom). (d) SCID mice with peritoneal tumors derived from ES-2 FLUC cells
were treated either with a pseudotype vector that targets lamr or a control pseudotype vector that targets LacZ for up to 10 days. Luciferase signal
intensity was imaged by IVIS before (day 1) and after (day 4, day 11) treatment with pseudotype vectors (left). Percentage of growth was quantified
for tumors treated with each pseudotype vector on day 4 and 11 (middle). Growth percentage is the amount of tumor growth compared to day 1.
Luciferase body counts from day 1, 4, and 11 were graphed to generate tumor growth curves (right) (*P < 0.05. **P < 0.001. ***P < 0.0001.).

Molecular Therapy vol. 18 no. 1 jan. 2010 71


LAMR as a Target for Novel Cancer Gene Therapy
the use of lentiviral vectors expressing siRNA-­targeting LAMR.43
These vectors were used for treatment and prevention of prion dis-
orders. To achieve specificity for tumor targeting, we developed a
novel method for targeting LAMR in vivo that utilizes both lenti-
viral vector shRNA expression as well as the tumor-targeting capa-
bilities of Sindbis viral vectors. We show that LAMR expression
levels can successfully be diminished through use of our Sindbis/
Lenti pseudotype vector and exposure of tumors to this vector
in vivo caused a reduction of tumor growth as ­predicted. Other
groups have used Sindbis/Lenti pseudotype vectors for different
purposes,44–46 some of which were based on our earlier Sindbis-
ligand-assisted targeting technology,47 but our studies are the first
to demonstrate the use of a Sindbis/Lenti pseudotype vector for
tumor-targeted knock down of gene expression in animal ­models.
By using unmodified Sindbis structural proteins, our pseudotype
vector maintains the natural tumor-targeting capabilities of Sindbis
virus through its receptor, LAMR. Thus our Sindbis/Lenti pseudo-
type vector can both target and inhibit tumor growth via binding/
downregulating LAMR.
Targeting LAMR in this study both affirms LAMR’s impor-
tance for cancer therapy and establishes our pseudotype vector as
a novel effective therapeutic agent. This represents a potentially
highly efficacious new therapeutic approach to cancer treatment,
which can utilize similar pseudotype vectors and approaches to
knock down other genes in vivo in a highly targeted manner.
Currently, Sindbis/Lenti-mediated reduction of LAMR is not
presently as effective as siRNA. We consistently observe ~50%
reduction in LAMR expression with our pseudotype vector
in vitro, whereas the siLAMR pool achieves ~90% reduction in
LAMR expression. This less potent reduction in LAMR expres-
sion is most likely due to the fact that the siRNA pool we use for
knocking down LAMR consists of four different oligonucleotide
sequences whereas our Sindbis/Lenti vector carries only one.
Generating pseudotype vectors that carry more than one shRNA
sequence may help increase its knock-down efficiency. In addition,
we have not yet been able to produce the Sindbis/Lenti pseudotype
vectors at as high a titer as we can achieve with Sindbis ­vectors
resulting in a transduction efficiencies that are not as high in vivo,
thus some tumor cells are not transduced and hence remain via-
ble. Further efforts to optimize the vectors described here will
develop more potent vectors targeting LAMR as well as delivery of
shRNA in vivo for additional therapeutic purposes. Finally, alter-
native strategies that target LAMR in a ribosomal context may
also be employed and our findings are supportive of the notion
that these efforts might ultimately be very advantageous. To this
end, our lab has recently solved the crystal structure of an LAMR
construct consisting of residues 1–220 (ref. 48) and are currently
using this structure as well as the system established by our cur-
rent results in pursuing the design of small molecules to inhibit
LAMR ­functions. It is likely that a combination of both vector-
and drug-based modalities will optimize treatment.
In conclusion, the studies reported here point to several mech-
anisms by which LAMR can contribute to tumor development;
further underscoring its importance as a target for cancer ther-
apy. In addition, we provide a novel mechanism for gene therapy
in vivo, which can be used against LAMR and potentially other
genes as well.
72
© The American Society of Gene & Cell Therapy
Materials and Methods
Protein analysis. For western blots, cells were harvested in Mammalian
Protein Extraction Reagent (Pierce, Rockford, IL). A 20 μg of total protein
was used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis
and transferred to polyvinylidene fluoride membrane. For LAMR protein
expression, we initially used the goat polyclonal antibody F-18 purchased
from Santa Cruz biotechnology (Santa Cruz, CA) (Figure 1). We later
switched to the rabbit polyclonal antibody H-141 (Santa Cruz) because of
its stronger signal. Both recognize epitopes on the C-terminus of LAMR
and predominantly detect 37-kd LAMR. The Survivin (A-19) antibody
was also purchased from Santa Cruz. The antibody for p21 is from BD
Biosciences (San Jose, CA), and β-actin (AC15) from Sigma-Aldrich
(St Louis, MO). All other cell-cycle antibodies were gifts from Dr Pagano
at the NYU School of Medicine. Western blots were quantified using The
NIH Image 1.63 program (Bethesda, MD).
To study translation, cells were metabolically labeled with 35S-
methionine (20 μCi/ml) (PerkinElmer, Waltham, MA) in Dulbecco’s
modified Eagle’s medium lacking methionine (MP Biomedicals, Santa
Ana, CA) supplemented with 0.1% fetal calf serum for 2 hours at 37 °C
(pulse). Normal growth medium was then added and cells were incubated
for an additional 90 minutes at 37 °C (chase). For trypsin treatement, cells
were incubated with 1× trypsin EDTA from cellgro Mediatech (Manassas,
VA), containing 0.05% trypsin and 0.53 mmol/l EDTA, for ~5 minutes to
allow cells to detach. Cells were then reseeded for labeling the next day.
Cells were harvested in Mammalian Protein Extraction Reagent and 20 μg
of protein was loaded onto sodium dodecyl sulfate–polyacrylamide gel
electrophoresis for analysis.
Gene analysis. Total RNA was extracted from cells using the RNA easy
mini kit (Qiagen, Valencia, CA) and its integrity was analyzed at the NYU
genomics core facility. RNA (0.5 μg) was used for reverse transcription
either with Reaction Ready first strand cDNA synthesis kit (SuperArray) or
iScript complementary DNA synthesis kit (BioRad, Hercules, CA). Human
LAMR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) prim-
ers were designed and used as previously described.36 Human GAPDH was
chosen as the housekeeping gene for comparative analysis. The fold change
in LAMR relative to the GAPDH endogenous control was determined by:
fold change = 2(–Ct), where CT = CT(LAMR) − CT(GAPDH) and (CT) = CT(ES-2/FLUC/
LRP)
− CT(ES-2/FLUC). CT is the threshold cycle determined for fluorescence data
collection. GAPDH and this formula were also used for analysis of cell
cycle–related genes. The cell-cycle RT2Profiler PCR array and primers for
validation of cyclin A2, cyclin B1, CDK2, E2F1, p21, Survivin, as well as an
additional GAPDH primer set were purchased from SuperArray. Primers
for CDK1 were obtained online from Primer Bank.49 Quantitative—real-
time PCR was performed with 0.5 μl of complementary DNA using a
BioRad iCycler.
Cell culture. HT1080, 293, HeLa, and HepG2, and ES-2 cells were obtained
from the ATCC (Manassas, VA). ES-2/FLUC cells were derived using a
pIRES2-Luc/EGFP plasmid as previously described.36 To ­generate HT1080
FLUC cells, the FLUC gene was amplified by PCR from the pIRES2-
Luc/EGFP plasmid and cloned into the TOPO pcDNA3.1v5/his vector
(Invitrogen, Carlsbad, CA) to create a pcDNA3.1/FLUCv5/his plasmid.
HT1080 cells were transfected with this plasmid and clones were selected
with neomycin. The predesigned siGENOME SMART pool ­targeting LAMR,
siGLO RISC-free siRNA, and DharmaFECT transfection reagents were
purchased from Dharmacon (Lafayette, CO). Individual oligo sequences
and the region of LAMR they target are provided (Supplementary Figure
S1). A final concentration of 100 nmol/l of siRNA was used for each
transfection. To determine siGLO transfection efficiency, nontransfected
and siGLO-transfected cells were analyzed 1 day after transfection on a
FACSCaliber machine. The Flowjo 8.2 program (Tree Star, Ashland, OR)
was used to quantify the percentage of siGLO-transfected cells positive in
the FL2-H channel, compared to nontransfected cells.
www.moleculartherapy.org vol. 18 no. 1 jan. 2010
© The American Society of Gene & Cell Therapy
Cells were imaged with a Nikon Eclipse TE200-E microscope
(Tokyo, Japan) using the NIS-Elements BR-2.30 program (Nikon). Cell
proliferation was measured using the CellTiter-Glo Cell Viability assay
from (Promega, Madison, WI) and luminescence was measured on a
GLOMAX 20/20 luminometer (Promega). For cell-cycle profile analysis,
cells were permeabilized overnight in 100% ethanol and then stained with
final concentrations of 50 μg/ml of propidium iodide and 100 μg/ml of
RNAse A (Sigma, St Louis, MO). Cells were then submitted to the NYU
Cancer institute core and analyzed on a FACSCaliber machine.
Ribosomal analysis. Ribosomes and polyribosomes were isolated and
analyzed as described by Rouquette et al.50 Briefly, siLAMR- and siGLO-
­transfected cells were treated with 100 μg/ml of cyclohexamide for 10
­minutes at 37 °C. An equal number of cells were pelleted and lysed in lysis
buffer with a Dounce homogenizer. Cell lysates were spun at 14,000 r.p.m.
for 10 minutes to remove cellular debris and then layered onto 10–50%
linear sucrose gradients (prepared ~16 hours before use). Gradients were
spun for 105 minutes at 40,000g in a SW41TI rotor (Beckman, Brea, CA).
A total of 24 (500-μl) fractions were collected from the top of each gradient
and optical density was measured at A260 nm. In addition, 32 μl of fractions
1–12 were added to 8 μl of 5× reducing buffer and used for western blot
analysis. Antibodies for ribosomal proteins L7a and S6 were purchased
from Cell Signaling (Danvers, MA).
Viral vector construction and production. To produce Sindbis/Lenti
pseudotype vectors, three packaging plasmids (pLP1, pLP2, and pLP/
E321), which provide structural proteins, and the expression plasmid
(pLenti6), encoding a modified lentiviral RNA genome, were used. Sindbis
viral envelope proteins (E3, E2, and E1) are provided by the pLP/E321
plasmid. To generate the pLP/E321 plasmid, the E321 gene fragment was
first amplified by PCR using pDHBB plasmid as a template, which contains
the full length of Sindbis structural proteins. The PCR product was first
cloned into pcDNA3.1 plasmid (Invitrogen) for sequencing validation and
was later used to replace the vesicular stomatitis virus-G fragment (from
StuI to PlmI site) in the pLP/VSVG plasmid.
pLenti6 carrying an ubiquitin C promoter (PUBC) was used to drive
Pol II-dependent expression of the firefly luciferase gene (pLenti6/FLUC).
A U6 shRNA promoter was used for shRNA expression (pLenti6/shLacZ
or pLenti6/shLAMR). The DNA oligos containing the corresponding
shRNA sequences against LacZ or LAMR genes were first cloned into the
pENTR/U6 plasmid and sequenced before recombination with pLenti6/
Block-iT-DEST plasmid according to manufacturer’s instruction.
Oligos used are as follows:
shLacZ top: 5′-CACCGCTACACAAATCAGCGATTTCGAAAAAT
CGCTGATTTGTGTAG-3′.
shLacZ bottom: 5′-AAAACTACACAAATCAGCGATTTTTCGAAA
TCGCTGATTTGTGTAGC-3′.
shLAMR top: 5′-CACCGCAACAAGGGAGCTCACTCACGAATG
AGTGAGCTCCCTTGTTG-3′.
shLAMR bottom: 5′-AAAACAACAAGGGAGCTCACTCATTCGT
GAGTGAGCTCCCTTGTTGC-3′.
Packaging plasmids (pLP1, pLP2, and pLP/E321) and a pLent6 plasmid
(pLenti6/FLUC, pLenti6/shLacZ, or pLenti6/shLAMR) were co-transfected
into 293T cells using the calcium phosphate method. Pseudotype vector
particles were collected in supernatant and pelleted using ultracentrifugation
at 25,000 r.p.m. for 2 hours with an SW-28 rotor. The vector pellets were
diluted into ×1/50 volume before centrifugation using Dulbecco’s modified
Eagle’s medium.
The capability of Sindbis/Lenti-shLAMR to suppress LAMR expression
was tested in culture using ES2/FLUC cells. ES2/FLUC cells were seeded onto
6-well plates the day before transduction. The next day, 100 μl virus plus 400 μl
of medium was added to the wells and incubated for 24 hours. The vector was
then removed and replaced with fresh medium and incubated for 48 hours.
Cells were lysed and LAMR expression determined by western blot.
Molecular Therapy vol. 18 no. 1 jan. 2010
LAMR as a Target for Novel Cancer Gene Therapy
Animals. For Sindbis/Lenti pseudotype–targeting experiments, ES-2
human ovarian carcinoma cells (2 million) were inoculated i.p. into female
SCID mice (4–8 weeks old, Taconic Farms, Hudson, NY). At 7, 8, and 9
days after injection of ES-2 cells, mice were treated with Sindbis/­Lenti-
FLUC vector (1 cm3, 106 plaque-forming units) by i.p. injection. At 10 days
after ES-2 injection, and 3 days of pseudotype vector treatment, mice were
subjected to IVIS imaging. For Sindbis/Lenti shLAMR experiments, ES2-
FLUC cells (3 million) were inoculated i.p. into female SCID mice (Taconic
Farms). One day after inoculation, first IVIS images were taken to establish
baseline tumor load. Mice were then treated daily i.p. with 0.5 ml of Sindbis/
Lenti-shLacZ or Sindbis/Lenti-shLAMR (~106 plaque-forming units) for
three consecutive days. After treatment, 4 days after ES-2 inoculation, mice
were subjected to a second IVIS imaging. Mice were then treated daily i.p.
with 0.5 ml of Sindbis/Lenti-shLacZ or Sindbis/Lenti-shLAMR (~106) for
four consecutive days, 7–10 days after ES-2 injection. At 11 days after ES-2
injection mice were subjected to a third IVIS imaging.
In vivo bioluminescence detection with the IVIS. A cryogenically cooled
IVIS Spectrum Imaging System (Caliper LifeScience, Alameda, CA) with
Living Image 3.0 acquisition and analysis software (version 2.11; Xenogen,
Alameda, CA) was used to detect the bioluminescence signals in mice.
Each mouse was injected i.p. with 0.3 ml of 15 mg/ml beetle luciferin
(potassium salt; Promega) in phosphate-buffered saline. After 5 minutes,
mice were anesthetized with isofluoran mixed oxygen (2%). The imaging
system first took a photographic image in the chamber under dim illumi-
nation, followed by luminescent image acquisition. Overlay of the pseudo-
color images represents the spatial distribution of photon counts produced
by active luciferase. An integration time of 1 minute at medium binning
was used for luminescent image acquisition for all animal tumor models.
Living Image 3.0 software was used to integrate the total bioluminescence
signals (in terms of photon counts) obtained from animals.
Statistical analysis. Data were analyzed using a standard Student’s t-test
using GraphPad Prism, version 3.0a for Macintosh (GraphPad Software,
San Diego, CA). All P values presented in this study are two-tailed. In
our analysis, results for experimental groups (siLAMR or shLAMR) were
considered statistically significant relative to the control group (siGLO or
shLacZ) if the P value <0.05.
Supplementary Material
Figure S1. siLAMR targeting sequences within the lamr coding region.
Acknowledgments
US Public Health grants CA100687 from the National Cancer Institute,
National Institutes of Health, and Department of Health and Human
Services supported this study. Funding was also provided by a gift
from the Litwin Foundation and a Research and License agreement be-
tween NYU and CynVec. Some of the authors have competing inter-
ests. Specifically, the contents of this study may be utilized for a patent.
According to the rules and regulations of New York University School of
Medicine, if this patent is licensed by a third party, some of the authors
may receive benefits in the form of royalties or equity participation.
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