[CANCER RESEARCH 63, 2716 –2722, June 1, 2003]
Advances in Brief
PRL-3 and PRL-1 Promote Cell Migration, Invasion, and Metastasis1,2
Qi Zeng,3 Jing-Ming Dong, Ke Guo, Jie Li, Hui-Xian Tan, Vicki Koh, Catherine J. Pallen, Edward Manser, and
Wanjin Hong
Institute of Molecular and Cell Biology, Singapore 117609, Singapore [Q. Z., J-M. D., K. G., J. L., H-X. T., V. K., E. M., W. H.]; Department of Pediatrics, University of British
Columbia, BC Research Institute for Children’s and Women’s Health, Vancouver, British Columbia V5Z 4H4, Canada [C. J. P.]
Abstract
We demonstrate here that Chinese hamster ovary cells stably expressing PRL-3, a Mr 20,000 prenylated protein tyrosine phosphatase, or its
relative, PRL-1, exhibit enhanced motility and invasive activity. A catalytically inactive PRL-3 mutant has significantly reduced migrationpromoting activity. We observe that PRL-3 is associated with diverse
membrane structures involved in cell movement. Furthermore, we show
that PRL-3- and -1-expressing cells, but not control cells, induce metastatic tumor formation in mice. Thus, our results deliver the first evidence
for a causative role of PRL-3 and -1 in promoting cell motility, invasion
activity, and metastasis.
Introduction
PRL-14 was originally identified as an immediate early gene of
which the transcript is induced during liver regeneration after partial
hepatectomy (1). Searching expression sequence tagged databases
with the PRL-1 sequence led us to identify PRL-2 and -3 (2). The
three members share 76 – 87% amino acid sequence identity. These
PRLs represent a novel class of PTP with a unique COOH-terminal
prenylation motif. Although PRL-1 was originally described as a
nuclear protein (1), our analysis of heterologously expressed PRL-1,
-2, and -3 has shown that these phosphatases are mainly associated
with the plasma membrane and endosomal structures in a prenylationdependent manner, and the majority of endogenous PRL-3 in intestine
epithelial cells is also non-nuclear (3). These data suggest that the
major sites of action of the mature, post-translationally prenylated
PRL-PTPs are at the membrane.
Many genes have been implicated in the development and progression of colorectal cancer (4, 5). However, the identification of consistent genetic alterations associated with the transition from the
primary tumors to metastatic colorectal liver disease has proved
elusive. Gene expression profiling revealed recently that among 144
up-regulated genes detected in metastatic colorectal liver samples,
PRL-3 is the only gene consistently overexpressed in all 18 of the
cancer metastases examined, with essentially undetectable PRL-3
expression in normal colorectal epithelia and intermediate expression
in advanced primary cancers (6). The overexpression of the PRL-3
transcript is because of gene amplification in 3 of 12 metastases
examined, whereas enhanced transcriptional activity likely accounts
Received 1/6/03; accepted 3/20/03.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by research grants from The Agency of Science, Technology and Research
(Aⴱ STAR), Singapore.
2
Supplementary data for this article are available at Cancer Research Online (http://
cancerres.aacrjournals.org).
3
To whom requests for reprints should be addressed, at Institute of Molecular and Cell
Biology, 30 Medical Drive, Singapore 117609, Singapore. Phone: 65-6874-3752; Fax:
65-6779-1117; E-mail: [email protected].
4
The abbreviations used are: PRL, phosphatase of regenerating liver; CHO, Chinese
hamster ovary; PTP, protein-tyrosine phosphatase; ␤-gal, ␤-galactosidase; FBS, fetal
bovine serum; EGFP, enhanced green fluorescent protein; PBSCM, PBS with CaCl2 and
MgCl2; TRITC, tetramethylrhodamine isothiocyanate; PTEN, phosphatase and tensin
homolog deleted in chromosome 10.
for elevated PRL-3 transcripts in the other metastases. This study
suggests the possibility that an excess of PRL-3 phosphatase is a key
alteration contributing to the acquisition of metastatic properties of the
tumor cells, but cell biological and mechanistic evidence supporting
such a role is lacking. In this study we provide evidence to support a
causal role of PRL-3 and -1 in tumor metastasis.
Materials and Methods
Generation of Stable CHO Cell Lines Expressing Myc-PRL-1, -3, and
␤-Gal. To introduce the Myc epitope (10 amino acid residues) at the NH2
termini of PRL-1 and -3, a PCR-based approach was used. Forward primer A
incorporating the Myc epitope (italicized; 5⬘-gc gaattc acc atg gag cag aag ctg
atc tcc gag gag gac ctc gct cga atg aac cgc cct gct c-3⬘) and reverse primer B
(5⬘-gt ggatcc tta ttg aat aca aca gtt g-3⬘) were used to amplify PRL-1 cDNA.
Forward primer E incorporating the Myc epitope (5⬘-gc gaattc acc atg gag cag
aag ctg atc tcc gag gag gac ctc gcc cgc atg aac cgg cct gcg cct g-3⬘) and
reverse primer F (5⬘-ct ggatcc cta cat gac gca gca tct ggt c-3⬘) were used to
amplify PRL-3 cDNA. The PCR fragments were digested with EcoRI and
BamHI, and inserted into the inducible expression vector pStar (7). CHO-K1
cells (American Type Culture Collection, Manassas, VA) were used to generate CHO cell lines stably expressing Myc-PRL-1 (clone 9), Myc-PRL-3
(clone 36), or ␤-gal (clone 8 and 13), as described previously (3, 7). Briefly,
the cells were transfected with the above pSTAR-Myc-PRL-1, Myc-PRL-3, or
␤-gal plasmids, and cultured in RPMI 1640 supplemented with 10% FBS and
selected in 1 mg/ml of neomycin. The c-Myc antibody (9E10) was from Santa
Cruz Biotechnology (Santa Cruz, CA).
Generation of CHO Cells Stably Expressing EGFP-PRL-3 and EGFPPRL-3 (C104S). Synthetic oligonucleotides were purchased from Oligos Etc.
(Wilsonville, OR). The Pyrococcus furiosus DNA polymerase was from Stratagene (La Jolla, CA). The pEGFP-C1 vector was from (Clontech).5 To prepare
a plasmid (pEGFP-PRL-3) for expression of PRL-3 NH2-terminally tagged
with EGFP (EGFP-PRL-3), forward primer 1 (5⬘-gtg aat tct atg gcc cgc atg aac
cgg-3⬘), and reverse primer F (above) were used to retrieve the PRL-3 coding
region by PCR. The PCR reaction was resolved by agarose gel electrophoresis,
and the specific fragment (530 bp) was eluted and purified. The fragment was
digested with EcoRI and BamHI, and then inserted into the corresponding sites
of the pEGFP-C1 vector. To construct pEGFP-PRL-3 (C104S) containing an
inactivating mutation of the essential catalytic cysteine residue to serine at
position 104 in the phosphatase active site, forward primer 1 in combination
with reverse primer 3 incorporating the desired nucleotide (C to G, italicized)
substitution (5⬘-cag gcc cgc cac aga gtg cac aag-3⬘) were used to retrieve the
coding region for the NH2-terminal portion of PRL-3 (N-fragment). Forward
primer 4 incorporating the desired nucleotide (G to C, italicized) substitution
(5⬘-ctt gtg cac tct gtg gcg ggc ctg-3⬘) and reverse primer F (above) were used
to retrieve the coding region for the COOH-terminal portion of PRL-3 (Cfragment). The gel-purified N-fragment and C-fragment were mixed and used
in a PCR reaction with forward primer 1 and reverse primer F to generate the
entire cDNA encoding the mutant PRL-3 (C104S). After digestion with EcoRI
and BamHI, the specific fragment (530 bp) was purified and cloned into the
corresponding sites of pEGFP-C1 vector. The two expression constructs were
confirmed by DNA sequencing and transfected into CHO-K1 (ATCC CCL-61)
cells using Lipofectamine 2000 (Invitrogen). The cells were cultured in RPMI
5
Internet address: http://www.clontech.com/index.shtml.
2716
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.
PRLs PROMOTE CELL MIGRATION, INVASION, AND METASTASIS
for 24 h. The confluent monolayer of cells was wounded as described in the
legend to Fig. 2, and the culture medium was then replaced with HEPESbuffered serum-free medium. Cell movement was monitored with a Nikon
inverted microscope using a ⫻40 oil lens. The microscope was equipped with
a homemade temperature control chamber set at 37°C. Time-lapse series of
images were captured using a Bio-Rad Radiance 2000 confocal system and
analyzed using LaserPix software (Bio-Rad).
Metastasis Assay. Ten-week old female nude mice (Jackson Labs) were
each injected via the tail vein with PRL-1-, -3-, or ␤-gal-expressing cells
(5 ⫻ 105). Mice were sacrificed 25 days after the tail vein injection, and all of
the tissues were examined for metastasis. Tissues with metastases were either
photographed for gross morphology or analyzed by H&E staining on freshfrozen sections.
Results
Fig. 1. PRL expression in CHO-Myc-PRL-1, CHO-Myc-PRL-3, and CHO-␤-gal stable
cell lines. A, lysates (25 ␮g) from the stable cell lines indicated at the top of the figure
were analyzed for PRL expression by immunoblotting with anti-PRL-1 rabbit serum and
were also probed with antibody to the Cdk inhibitor p27Kip1 as a loading control. The
anti-PRL-1 serum cross-reacts with PRL-3 (Lane 3). B, the lysates were independently
probed with anti-PRL-3 rabbit serum, which cross-reacts with PRL-1 (Lane 2). C, cell
lysates were probed with mouse anti-Myc antibody 9E10 to detect Myc-tagged PRL-1 and
-3, and also probed with anti-RhoGDI1 rabbit antibody as a loading control. Endogenous
PRLs were not detected in the ␤-gal clone 13 (Lane 1) or clone 8 (data not shown).
1640 supplemented with 10% FBS and selected in 1 mg/ml of neomycin for
20 –30 days to establish stable cell pools.
Cell Motility Assay. This was performed as described (8) using Transwells
(6.5 mM diameter; 8 ␮M pore size polycarbonate membrane) obtained from
Corning.6 Cells (1 ⫻ 105) in 0.5 ml serum-free medium were placed in the
upper chamber, whereas the lower chamber was loaded with 0.8 ml medium
containing 10% FBS. The total number of cells that migrated into the lower
chamber was counted after 24 h of incubation at 37°C with 5% CO2.
Cell Invasion Assay. This was carried out essentially as described (8, 9).
Transwells (BD Biocoat Matrigel 24-well invasion chamber) with filters
coated with extracellular matrix (ECMatrix gel) on the upper surface were
obtained from BD Biosciences.7 The experiments were performed according to
the manufacturer’s protocol. Cells (1.84 ⫻ 105) were added to the upper
chamber in serum-free medium containing 0.1% BSA, and the total invasive
cells in the lower chamber were counted after 48 h of incubation at 37°C with
5% CO2.
Confocal Microscopy. The pools of cells stably expressing EGFP-PRLs
were seeded onto glass coverslips and grew for 24 h. Cells were washed twice
with PBSCM and then fixed in 3% paraformaldehyde for 20 min at room
temperature. After three more washes with PBSCM, the cells were permeabilized for 15 min with 0.1% saponin in the same buffer. Cells were washed three
times with PBSCM and incubated with TRITC-conjugated Phalloidin (Molecular Probes) for 1 h. The cells were washed four times with PBSCM and
mounted onto a glass slide with one drop of antifade reagent in PBS glycerol
(Biomedia Corp., Foster City, CA), and kept at 4°C in the dark until analysis.
Confocal imaging was performed using a laser scanning head (MRC 1024;
Bio-Rad Laboratories, Hertfordshire, United Kingdom).
Live Cell Imaging. Cells (5 ⫻ 105) were seeded onto each 35-mm glass
bottom dish (MatTek Co., Ashland, MA) and cultured at 37°C with 5% CO2
6
7
Internet address: http://www.corning.com.
Internet address: http://www.bdbiosciences.com/index.shtml.
Myc-PRL-3 and Myc-PRL-1 Enhance Cell Motility and Invasive Activity. We have previously established stable CHO cell lines
expressing Myc-tagged PRL-1 (Myc-PRL-1, clone 9), Myc-tagged
PRL-3 (Myc-PRL-3, clone 36), and as a negative control, ␤-gal
(␤-gal, clones 8 and 13). The ␤-gal-expressing CHO cells have no
detectable endogenous PRL expression (Fig. 1, A and B). Heterologous protein expression was achieved by transfection of the cells with
the tetracycline-responsive pStar vector containing the appropriate
cDNA (3, 7). Tetracycline-enhanced overexpression of Myc-PRL-1 or
-3 was toxic to the cells, but a significant level of Myc-PRL-1 or -3
was constitutively expressed in these stable cell lines without tetracycline induction (Fig. 1C). Thus, we focused on determining the
effect of the constitutive levels of Myc-PRL-1 and -3 expression on
cell migration and invasion. When cell motility was examined using
the standard Transwell assay (8), ⬃5-fold more Myc-PRL-1- and
-3-expressing cells migrated to the bottom chamber than did the
control ␤-gal-expressing cells (Fig. 2A). The invasive capacities of the
cells were assessed using Transwells with filters coated with extracellular matrix gel (8, 9), and were found to be enhanced ⬃8-fold by
Myc-PRL-1 or -3 expression compared with control ␤-gal-expression
Fig. 2. Myc-PRL-1 or Myc-PRL-3 expression enhances cell motility and invasiveness.
A, the migration of stable CHO cell lines expressing ␤-gal, Myc-PRL-1, or Myc-PRL-3
was quantitated over 24 h using the Transwell assay. B, the invasive abilities of the same
cell lines were examined over 48 h using Transwells with filters coated with extracellular
matrix. In both A and B, the bars represent the mean of three independent experiments;
bars, ⫾SD. Three separate Transwells were used in each independent experiment for each
cell line.
2717
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.
PRLs PROMOTE CELL MIGRATION, INVASION, AND METASTASIS
Fig. 3. Enhanced motility of Myc-PRL-1- and Myc-PRL-3-expressing cells in wound-healing assays. A, monolayers of confluent Myc-PRL-1-, Myc-PRL-3-, and ␤-gal-expressing
cells were wounded with yellow pipette tips. After washing with warm PBS, the cells were incubated in fresh culture medium. The wounded areas were photographed at the beginning
(0 h, top panels) and the end (5 h, bottom panels) of the assay. Bar, 100 ␮m. B. images are shown from the start (0 min, a and c) and end (400 min, b and d) of the time-lapse videos
of Myc-PRL-3-expressing cells (a and b, from video 1) and ␤-gal-expressing cells (c and d, from video 2) monitored in wound-healing assays. The black lines demarcate the original
wound edge. Tracked cells are labeled as T1 to T9. The migration paths taken by these cells are indicated with yellow lines. Bars, 50 ␮m. C, the velocities of migration of the tracked
cells are shown. Brackets indicate the number of cells used for velocity measurements.
(Fig. 2B). To confirm these observations, cell motility was independently assessed by wound-healing assays (10). As shown in Fig. 3A,
Myc-PRL-1- and -3-expressing cells migrated much faster than ␤-galexpressing cells. The Myc-PRL-expressing cells migrated a ⬃5-cell
distance, whereas the ␤-gal control cells migrated only ⬃1-cell distance during a 5-h incubation after wounding. Thus, Myc-PRL-3 and
-1 cells have greatly enhanced motility and invasive ability as compared with control cells. To more exactly measure the velocity of cell
motility for these cell lines, live cell time-lapse imaging analysis was
performed on Myc-PRL-3 cells (Supplementary Data, video 1) and
control ␤-gal cells (Supplementary Data, video 2) for 400 min. Images
obtained at the start and end of this time period are shown in Fig. 3B.
The front line of the Myc-PRL-3 cell sheet at the wound edge moved
much faster than did that of control ␤-gal cells. The movement of
individual cells at the leading edge was tracked manually using
LaserPix software (Bio-Rad). The velocities of Myc-PRL-3-expressing cells and control ␤-gal-expressing cells were calculated, respectively, as 14.70 ⫾ 4.41 ␮m/h and 5.04 ⫾ 2.08 ␮m/h (P ⬍ 0.00001,
by Student’s t test; Fig. 3C).
EGFP-PRL-3 Is Enriched at Plasma Membrane Structures
Involved in Cell Movement. Our previous study has shown that
Myc-PRL-1, -2, and -3 are distributed to the plasma membrane and
endosomal compartments (3). In view of the enhanced migration and
invasion properties of PRL-1- and -3-expressing cells, we more
closely examined the localization of PRL-3 on the cell surface using
a pool of stable-transfected CHO cells expressing EGFP-tagged
2718
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.
PRLs PROMOTE CELL MIGRATION, INVASION, AND METASTASIS
Fig. 4. EGFP-PRL-3 associates with various
membrane structures involved in cell movement.
CHO cells expressing EGFP-PRL-3 were processed for fluorescence microscopy without (a– c)
or with labeling with TRITC-conjugated phalloidin
(d–f). EGFP-PRL-3 was enriched in membrane
processes such as ruffles (a, d, and e, long arrows),
membrane protrusions (a and b, short arrows), and
vacuolar-like and semivacuolar-like membrane extensions (c, arrowheads). These cells were also
processed for labeling with TRITC-conjugated
phalloidin (Molecular Probes) to detect the distribution of endogenous actin filaments. EGFPPRL-3 (d), phalloidin-labeled actin (e), and the
merged image (f) are shown. Bar, 10 ␮m.
PRL-3. The EGFP-PRL-3 was enriched in several membrane processes on the cell surface, including ruffles, protrusions, and some
vacuolar-like membrane extensions (Fig. 4). These structures have
been demonstrated to play a role in cell movement (11).
Catalytically Inactive PRL-3 Has Significantly Reduced
Migration-promoting Activity. To probe the molecular basis for
PRL-3-enhanced cell motility and invasion, we next examined
whether its catalytic activity as a phosphatase is coupled to an ability
to promote cell migration. We created a mutant form of EGPF-PRL-3,
EGPF-PRL-3 (C104S), by mutating the essential Cys104 of the phosphatase active site signature motif to Ser. This active site cysteine is
invariant among all of the PTPs (12), and without exception and
including PRL-3 (13), mutation of this residue to serine or alanine
abolishes PTP activity (12, 14). A pool of stably transfected CHO
cells expressing EGFP-PRL-3 (C104S) was established. Using live
cell imaging, the intracellular distribution of EGFP-PRL-3 and EGPFPRL-3 (C104S), and the migration behavior of the cells were studied
during 4-h wound-healing assays. The first and the last images of the
series are shown in Fig. 5, A (fluorescence) and B (phase contrast).
Examination of EGFP-PRL-3 localization showed that it was concentrated at membrane structures (ruffles and protrusions; Fig. 5A) that
were dynamic during cell movement (Supplementary Data, video 3).
The EGFP-PRL-3 (C104S) was similarly associated with membrane
structures (Fig. 5A; Supplementary Data, video 4); however, reduced
motility was noted in EGFP-PRL3 (C104S) -expressing cells as
compared with EGFP-PRL-3-expressing cells. By tracking individual
cells, the average calculated velocities for EGFP-PRL-3 and EGFPPRL-3 (C104S) cells were 24.01 ⫾ 3.55 ␮m/h and 12.86 ⫾ 4.99
␮m/h, respectively (P ⬍ 0.0005, Student’s t test; Fig. 5C).
PRL-3- and PRL-1-expressing Cells, But Not Control Cells,
Induce Metastatic Tumor Formation in Nude Mice. Key events in
metastasis include the ability of tumor cells to survive in and then
leave the circulation to extravagate into a tissue, begin and maintain
growth in this tissue to form preangiogenic micrometastases, and then
develop a blood supply that enables formation of macroscopic tumors
(15–17). We used an experimental metastasis assay to examine the
abilities of the PRL-expressing cells to carry out this process in vivo.
Five ⫻ 105 cells from Myc-PRL-1, -3, and control ␤-gal clones were
respectively injected into the tail veins of 10-week-old female nude
mice, thus introducing these cells directly into the circulatory blood
system of the animals. One of the mice carrying PRL-3-expressing
cells died on day 25 after injection and was found to have had an
extensive lung metastasis. At this time, examination of all of the
tissues in these mice revealed that 10 of 10 mice injected with
PRL-1-expressing cells had lung tumors (Fig. 6). Ten of 10 mice
carrying PRL-3-expressing cells had more extensive lung tumors; in
addition, 2 of them had liver metastasis tumors (Fig. 6). In contrast, no
tumors were found in 10 mice injected with ␤-gal-expressing cells
(Fig. 6). Human colon cancer cells are taken by the hepatic-portal
circulatory system first to the liver, the primary site of metastasis. In
our experimental metastasis assay, cells injected into the tail vein
initially arrive in the lung.
Discussion
Tumor cell metastasis is responsible for most cancer deaths (15–
17). However, the specific molecular changes in a tumor cell that
promote the metastatic process are largely unclear. The recent discovery that PRL-3 is the only gene consistently overexpressed in
100% of 18 colorectal cancer liver metastases examined suggests that
PRL-3 is a key factor involved in metastasis of this and possibly other
types of tumors (6). This striking report prompted us to directly test
whether stable expression of the protein tyrosine phosphatase PRL-3
and its relative PRL-1 in nonmetastatic CHO cells could induce the
acquisition of metastasis-associated properties such as enhanced invasiveness and motility, and confer metastatic ability. Greatly enhanced motilities of Myc-PRL-3- and -1-expressing cells were indeed
observed using two independent assays. Firstly, quantitative Transwell-based assays showed that PRL-3 and -1 expression enhanced
cell migration by ⬃5-fold. Secondly, qualitative wound-healing assays and live-cell imaging studies supported this conclusion. Furthermore, the velocity of migration of Myc-PRL-3 expressing cells was
⬃3-fold higher than the ␤-gal-expressing control cells. In addition to
increased motility, the Myc-PRL-3- and Myc-PRL-1-expressing cells
exhibited an ⬃8-fold increase in invasive abilities over the control
␤-gal-expressing CHO cells as assessed by an ECMatrix gelmembrane coated Transwell assay. As CHO cells have no detectable
endogenous PRL expression (Fig. 1), the crucial event that induced
2719
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.
PRLs PROMOTE CELL MIGRATION, INVASION, AND METASTASIS
Fig. 5. PRL-3 motility-enhancing activity is
coupled to its phosphatase active site. Equivalent
numbers (5 ⫻ 105) of EGFP-PRL-3 and EGFPPRL-3 (C104S) -expressing stable cell pools were
seeded, wounded 24 h later, and then videorecorded at 1 frame per 1 min over a period of 4 h.
A, images of pooled cells stably expressing EGFPPRL-3 from the start (0 min) and end (240 min) of
time lapse video 3 are shown in a and b. Images of
pooled cells stably expressing inactive mutant
EGFP-PRL-3 (C104S) from the start (0 min) and
end (240 min) of time-lapse video 4 are shown in c
and d. Note that only cells strongly expressing
EGFP-PRL or its mutant form are apparent in these
images. Both EGFP-PRL-3 and EGFP-PRL-3
(C104S) are associated with various membrane
processes during cell migration. However, EGFPPRL-3 is associated with more dynamic plasma
membrane structures during cell migration (videos
3 and 4). Tracked cells are represented as T1 to
T10. The migration paths taken by these cells are
indicated with red lines. Bar, 20 ␮m. B, phase
contrast images show equivalent densities of the
plated EGFP-PRL-3 and EGFP-PRL-3 (C104S)
-expressing cell pools. The black lines demarcate
the edges of the cell monolayer at 0 and 240 min.
Bar, 50 ␮m. C, the velocities of migration of the
tracked cells are shown for both stable pools.
Brackets indicate the number of cells used for velocity measurements.
the gain of motile and invasive properties by the CHO cells was the
expression of PRL-1 or -3.
This conclusion was additionally strengthened and extended by our
studies of independently and differently generated CHO cells stably
expressing wild-type active PRL-3 or a catalytically inactive PRL-3
(C104S) mutant. In this system, PRL-3 was tagged with EGFP instead
of Myc and expressed using the pEGFP-C1 instead of the pSTAR
plasmid vector. Also, pools of these stably transfected cells were used
for analysis to avoid clonal variations. Notably, EGFP-PRL-3 expression dramatically enhanced cell migration, whereas mutant EGFPPRL-3 (C104S) expression had a greatly reduced effect on promoting
cell migration, suggesting that the phosphatase activity of PRL-3 is
required for optimal PRL-3-dependent migration. The CHO-EGFPPRL-3 cell pool exhibited a higher velocity of migration than the
CHO-Myc-PRL-3 cell line (24.01 ⫾ 3.55 versus 14.70 ⫾ 4.41 ␮m/h,
respectively). This could be because of an inherent effect of EGFP on
2720
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.
PRLs PROMOTE CELL MIGRATION, INVASION, AND METASTASIS
Fig. 6. The expression of Myc-PRL-1 and Myc-PRL-3 promotes cell metastasis in vivo. Mice were examined 25 days after tail vein injection of (5 ⫻ 105) Myc-PRL-1-, Myc-PRL-3-,
or ␤-gal-expressing (clone13) cells. Metastatic tumors were found in the lungs of all of the mice injected with Myc-PRL-1 and Myc-PRL-3 cells, whereas 2 of the 10 mice injected
with Myc-PRL-3 cells also developed liver metastases. The top panels show the gross morphology of the respective lungs and/or liver, whereas the bottom panels show the histological
morphologies of sections derived from the respective tissues and stained with H&E. T stands for areas with tumor. Bars, 2.5 mm and 100 ␮m for the top and bottom panels, respectively.
the cells. Alternatively, it may be because of different PRL-3 expression levels driven by different promoters (cytomegalovirus promoter
of pEGFP-C1 versus the uninduced tetracycline-inducible operator
mini promoter of pSTAR) or gene copy numbers. In support of this,
we observed that cells with different EGFP-PRL-3 expression levels
showed different morphology and motility: the higher the expression
levels, the faster the cells moved. Also, the PRL-3 gene was found in
multiple copies within a small amplicon in 25% of actual colorectal
cancer liver metastases (6), indicating that gene amplification is an
important mechanism to effect elevated PRL-3 expression in metastases. Interestingly, our independent experiments revealed a difference in migration velocity between the CHO-EGFP-PRL-3 (C104S)
cell pool and the control CHO-␤-gal cell line (12.86 ⫾ 4.99 versus
5.04 ⫾ 2.08 ␮m/h, respectively), either because of an inherent EGFP
effect or raising the possibility that high levels of EGFP-PRL-3
(C104S) expression could enhance cell motility in a phosphataseindependent manner. Nevertheless, both Myc-PRL-3- and EGFPPRL-3-expressing cells demonstrated enhanced cell motility compared with their respective ␤-gal- or EGFP-PRL-3- (C104S)
expressing control cells by 3-fold and 2-fold.
The rapid metastasis in our animal study suggests that PRL-3 and
-1 can act as key players to initiate and maintain tumor cell growth in
a “foreign territory.” Examination of the expression levels of PRL-1,
-2, and -3 in other metastatic tumors will provide insight regarding the
possibility that these PRL-PTPs, and perhaps PRL-2, could be associated in general with metastatic events. The discovery that PRL-3 is
overexpressed in all of the examined metastatic colorectal cancers (6)
indicates that PRL-3 is a potential molecular marker for clinical
estimation of tumor aggressiveness. This, together with our present
study demonstrating that PRL-3 overexpression promotes cellular
properties associated with metastasis as well as the end point formation of macroscopic tumors in an in vivo metastasis assay, suggest that
PRL-3 may additionally present an excellent target for intervention
with colorectal tumor metastasis. This could be an important new
therapeutic opportunity, because most of the described genetic alterations in colorectal cancers involve the inactivation of tumor suppres-
sor genes, which are difficult to target with drugs (18, 19). Because
optimal PRL-3-enhanced cell migration is dependent on preservation
of its catalytic function, the consensus phosphatase motif will potentially be a therapeutic target. Also, the prenylation-dependent association of the PRL-PTPs with cell membranes (3), coupled with the
present description of the association of PRL-3 with membrane structures including ruffles, protrusions, and some vacuolar-like membrane
extensions could represent another opportunity for intervention. These
membrane structures have been demonstrated to play a role in cell
movement and invasion (10, 11). The expression of membrane-associated PRL-3 may induce dephosphorylation of target substrates at the
cell membrane to modulate the organization of the plasma membrane
in such a way to promote cell motility and invasion. No protein
substrate has yet been identified for PRL-3, and classic in vitro
phosphatase substrates are poorly dephosphorylated by recombinant
PRL-3.8 This latter property is similar to PTEN, a 3⬘-lipid phosphatase of phosphoinositides (20). Because PRL-1, -2, and -3 share some
homology with PTEN in the phosphatase active site “signature motif”
and flanking regions (1), one might speculate that PRL-3, like PTEN,
acts as a lipid phosphatase at the cytoplasmic face of the plasma
membrane. Our data raise the possibility that disruption of this prenylation-dependent association could effectively block the function(s)
of PRL-3 in cell migration and invasion. In this regard, we have
shown previously that treatment of cells with the prenyltransferase
inhibitor FTI-277 (which specifically inhibits protein farnesylation;
Ref. 21) induces the subcellular redistribution of PRLs from plasma
membrane to the nucleus (3). Notably, some reports support the
potential use of farnesyltransferase inhibitors as antimetastatic agents
in cancer therapy (22, 23). It will be of considerable interest to
determine whether such inhibitors of prenylation affect PRL-3- or
PRL-1-dependent cell migration, invasiveness, and metastasis in our
cell model system. In conclusion, the viability of PRL-3 as an antimetastatic target warrants assessment.
8
Unpublished observations.
2721
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.
PRLs PROMOTE CELL MIGRATION, INVASION, AND METASTASIS
Acknowledgments
We thank Drs. Bor Luen Tang, Xin-Min Cao, and Xiao Hang Yang for
critical reading of the manuscript, and Drs. Yin Hwee Tan and Louis Lim for
their support.
References
1. Diamond, R. H., Cressman, D. E., Laz, T. M., Abrams, C. S., and Taub, R. PRL-1,
a unique nuclear protein tyrosine phosphatase, affects cell growth. Mol. Cell. Biol.,
14: 3752–3762, 1994.
2. Zeng, Q., Hong, W., and Tan, Y. H. Mouse PRL-2 and PRL-3, two potentially
prenylated protein tyrosine phosphatases homologous to PRL-1. Biochem. Biophys.
Res. Commun., 244: 421– 427, 1998.
3. Zeng, Q., Si, X. N., Horstmann, H., Xu, Y., Hong, W., and Pallen, C. J. Prenylationdependent association of protein tyrosine phosphatases PRL-1, -2, and -3 with the
plasma membrane and the early endosome. J. Biol. Chem., 275: 21444 –21452, 2001.
4. Kinzler, K. W., and Vogelstein, B. Lessons from hereditary colorectal cancer. Cell,
87: 159 –170, 1996.
5. Hanahan, D., and Weinberg, R. A. The hallmarks of cancer. Cell, 100: 57–70, 2000.
6. Saha, S., Bardelli, A., Buckhaults, P., Velculescu, V. E., Rago, C., St Croix, B.,
Romans, K. E., Choti, M. A., Lengauer, C., Kinzler, K. W., and Vogelstein, B. A
phosphatase associated with metastasis of colorectal cancer. Science (Wash. DC),
294: 1343–1346, 2001.
7. Zeng, Q., Tan, Y. H., and Hong, W. A single plasmid vector (pSTAR) mediating
efficient tetracycline-induced gene expression. Anal. Biochem., 259: 187–194, 1998.
8. Nguyen, D. H. D., Hussaini, I. M., and Gonias, S. L. Binding of urokinase-type
plasminogen activator to its receptor in MCF-7 cells activates extracellular signalregulated kinase 1 and 2 which is required for increased cellular motility. J. Biol.
Chem., 273: 8502– 8507, 1998.
9. Clark, E. A., Golub, T. R., Lander, E. S., and Hynes, R. O. Genomic analysis of
metastasis reveals an essential role for RhoC. Nature (Lond.), 406: 532–535, 2000.
10. Nobes, C. D., and Hall, A. Rho GTPases control polarity, protrusion, and adhesion
during cell movement. J. Cell Biol., 144: 1235–1244, 1999.
11. Small, J. V., Stradal, T., Vignal, E., and Rottner, K. The lamellipodium: where
motility begins. Trends Cell Biol., 12: 112–120, 2002.
12. Andersen, J. N., Mortensen, O. H., Peters, G. H., Drake, P. G., Iversen, L. F., Olsen,
O. H., Jansen, P. G., Andersen, H. S., Tonks, N. K., and Moller, N. P. H. Structural
and evolutionary relationships among protein tyrosine phosphatase domains. Mol.
Cell. Biol., 21: 7117–7136, 2001.
13. Matter, W. F., Estridge, T., Zhang, C., Belagaje, R., Stancato, L., Dixon, J., Johnson,
B., Bloem, L., Pickard, T., Donaghue, M., Acton, S., Jeyaseelan, R., Kadambi, V., and
Vlahos, C. J. Role of PRL-3, a human muscle-specific tyrosine phosphatase, in
angiotensin-II signaling. Biochem. Biophys. Res. Commun., 283: 1061–1068, 2001.
14. Guan, K., and Dixon, J. E. Evidence for protein-tyrosine-phosphatase catalysis
proceeding via a cysteine-phosphate intermediate. J. Biol. Chem., 266: 17026 –17030,
1991.
15. Chambers, A. F., Groom, A. C., and MacDonal, I. C. Dissemination and growth of
cancer cells in metastatic sites. Nat. Rev. Cancer., 2: 563–572, 2002.
16. Takeda, A., Stoeltzing, O., Ahmad, S. A., Reinmuth, N., Liu, W., Parikh, A., Fan, F.,
Akagi, M., and Ellis, L. M. Role of angiogenesis in the development and growth of
liver metastasis. Ann. Surg. Oncol., 9: 610 – 616, 2002.
17. Weiss, L. Metastasis of cancer: a conceptual history from antiquity to the 1990s.
Cancer Metastasis Rev., 19: 193–383, 2000.
18. Marx, J. Cancer research. New insights into metastasis. Science (Wash. DC), 294:
281–282, 2001.
19. Vogelstein, B., Lane, D., and Levine, A. J. Surfing the p53 network. Nature (Lond.),
408: 307–310, 2000.
20. Maehama, T., Taylor, G. S., and Dixon, J. E. PTEN and myotubularin: novel
phosphoinositide phosphatases. Annu. Rev. Biochem., 70: 247–279, 2001.
21. Lerner, E. C., Qian, Y., Blaskovich, M. A., Fossum, R. D., Vogt, A., Sun, J., Cox,
A. D., Der, C. J., Hamilton, A. D., and Sebti, S. M. Ras CAAX peptidomimetic
FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes. J. Biol. Chem., 270: 26802–26806, 1995.
22. Ohkanda, J., Knowles, D. B., Blaskovich, M. A., Sebti, S. M., and Hamilton, A. D.
Inhibitors of protein farnesyltransferase as novel anticancer agents. Curr. Top. Med.
Chem., 2: 303–323, 2002.
23. Andela, V. B., Rosenblatt, J. D., Schwarz, E. M., Puzas, E. J., O’Keefe, R. J., and
Rosier, R. N. Synergism of aminobisphosphonates and farnesyl transferase inhibitors
on tumor metastasis. Clin. Orthop., 397: 228 –239, 2002.
2722
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.
PRL-3 and PRL-1 Promote Cell Migration, Invasion, and
Metastasis
Qi Zeng, Jing-Ming Dong, Ke Guo, et al.
Cancer Res 2003;63:2716-2722.
Updated version
Supplementary
Material
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/63/11/2716
Access the most recent supplemental material at:
http://cancerres.aacrjournals.org/content/suppl/2003/06/03/63.11.2716.DC1
This article cites 20 articles, 8 of which you can access for free at:
http://cancerres.aacrjournals.org/content/63/11/2716.full#ref-list-1
This article has been cited by 43 HighWire-hosted articles. Access the articles at:
http://cancerres.aacrjournals.org/content/63/11/2716.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.