Podocalyxin-like 1 promotes invadopodia formation and metastasis through activation of Rac1/Cdc42/cortactin signaling in breast cancer cells
Advance Access publication June
Podocalyxin-like 1 promotes invadopodia formation and metastasis through activation of Rac1/Cdc42/cortactin signaling in breast cancer cells
Cheng-Wei Lin 1 2
Min-Siou Sun 0 1
Mei-Ying Liao 1
Chu-Hung Chung 1
Yi-Hsuan Chi 1
Li-Tin Chiou 1
John Yu 1 3
Kuo-Lung Lou 0
Han-Chung Wu 0 1 3
0 Graduate Institute of Oral Biology, School of Dentistry, College of Medicine, National Taiwan University , Taipei 106 , Taiwan
1 Institute of Cellular and Organismic Biology, Academia Sinica , Taipei 115 , Taiwan
2 Department of Biochemistry, School of Medicine, Taipei Medical University , Taipei 110 , Taiwan
3 Genomics Research Center , Academia Sinica, Taipei 115 , Taiwan
Metastatic disease is the leading cause of cancer mortality. Identifying biomarkers and regulatory mechanisms is important toward developing diagnostic and therapeutic tools against metastatic cancer. In this study, we demonstrated that podocalyxin-like 1 (PODXL) is overexpressed in breast tumor cells and increased in lymph node metastatic cancer. Mechanistically, we found that the expression of PODXL was associated with cell motility and invasiveness. Suppression of PODXL in MDA-MB-231 cells reduced lamellipodia formation and focal adhesion kinase (FAK) and paxillin phosphorylation. PODXL knockdown reduced the formation of invadopodia, such as inhibiting the colocalization of F-actin with cortactin and suppressing phosphorylation of cortactin and neural Wiskott-Aldrich syndrome protein. Conversely, overexpression of PODXL in MCF7 cells induced F-actin/cortactin colocalization and enhanced invadopodia formation and activation. Invadopodia activity and tumor invasion in PODXLknockdown cells are similar to that in cortactin-knockdown cells. We further found that the DTHL motif in PODXL is crucial for regulating cortactin phosphorylation and Rac1/Cdc42 activation. Inhibition of Rac1/Cdc42 impeded PODXL-mediated cortactin activation and FAK and paxillin phosphorylation. Moreover, inhibition of PODXL in MDA-MB-231 cells significantly suppressed tumor colonization in the lungs and distant metastases, similar to those in cortactin-knockdown cells. These findings show that overexpression of PODXL enhanced invadopodia formation and tumor metastasis by inducing Rac1/Cdc42/cortactin signaling network.
Tumor metastasis is the leading cause of cancer mortality;
identifying genes involved in tumor migration/invasion is paramount to the
development of effective treatments. Degradation of the
extracellular matrix (ECM) by tumor cells is an important step in tumor
invasion and metastasis. Tumor cells secrete proteases that degrade ECM
components. In addition, formation of invadopodia allows cells to
coordinate ECM degradation, thereby facilitating cell migration and
). Invadopodia are actin-rich membrane protrusions
driven by clusters of intracellular components, which include focal
adhesion kinase (FAK), paxillin, neural Wiskott–Aldrich syndrome
protein (N-WASp) and members of the Rho GTPase family, such as
Abbreviations: ECM, extracellular matrix; FAK, focal adhesion kinase; GFP,
green fluorescent protein; NOD/SCID, non-obese diabetic/severe combined
immunodeficiency; N-WASp, neural Wiskott–Aldrich syndrome protein;
PODXL, podocalyxin-like 1.
Cdc42 and Rac1. Activation and/or phosphorylation of these
molecules promote invadopodia formation (
). Cortactin plays a
crucial role in orchestrating activation of actin-nucleating factors and
actin assembly during invadopodia formation. Cortactin binds to
filamentous (F)-actin and facilitates the binding of other actin
regulatory proteins, including N-WASp and Arp2/3, which further
supports nucleation of a branched F-actin network (
). Expression of
cortactin promotes cell motility by enhancing ECM secretion and the
persistence of lamellipodia, a second type of actin-based protrusion,
which are localized in the leading edge of motile cell and act in cell
). The association between cortactin and invadopodia
was demonstrated to play a critical role in tumor invasiveness and
Podocalyxin-like 1 (PODXL), also known as Gp135, was initially
identified in podocytes of renal glomeruli that are instrumental in
kidney development (
). The structure of PODXL is closely related to
CD34 and endoglycan, a sialomucin in the plasma membrane (
Expression of PODXL was identified in podocytes, hematopoietic
progenitors, vascular endothelia and embryonic stem cells (
). It has
been reported that PODXL disrupts cell–junction complex
localization and decreases tight junction-dependent transepithelial resistance
). In addition, the association of PODXL with actin-binding
proteins such as Na+/H+ exchanger regulatory factors and Ezrin was
reported to be involved in the antiadhesive and migratory capabilities
of tumor cells (
). Recent studies showed that increased
expression of PODXL is correlated with poor prognoses in certain types
of cancer (
), in particular, high expression of PODXL is
associated with lymphatic invasion in breast cancers (28). Although the
expression of PODXL has been reported to promote antiadhesive and
migratory abilities, whether PODXL participates in tumor metastases
remains unclear. Hence, the role of PODXL and its detail regulatory
mechanism in tumor metastasis still need to be elucidated.
In the current study, we provide evidence demonstrating the critical
functional role of PODXL in promoting invadopodia formation and
tumor metastases. PODXL was found to induce
Rac1/Cdc42/cortactin signaling cascade and to regulate invadopodia formation, leading
to promotion of tumor invasiveness and metastasis.
Materials and methods
The human breast cancer cell lines MDA-MB-231 and MCF-7 were
purchased from Bioresource Collection and Research Center (Hsinchu,
Taiwan). MCF-7 cells were grown in modified Eagle medium supplemented
with non-essential amino acid and sodium pyruvate. MDA-MB-231 cells
were grown in Dulbecco’s modified Eagle medium-F12. All culture media
were supplemented with 10% fetal bovine serum (Gibco) and penicillin/
streptomycin (Gibco) and were grown at 37°C in a 5% CO2 atmosphere.
Cells were cultured and stored according to the suppliers’ instructions
and used at passages 10 to 20. Once resuscitated, cell lines were routinely
authenticated through cell morphology monitoring, growth curve analysis
and identity verification using short tandem repeat profiling analysis to
ensure no contamination.
Full-length human PODXL complementary DNA (PODXL-wt) was obtained
from Origene (Rockville, MD) and was subcloned into the pCMV6-DDK
vector. PODXL-related mutants (PODXL-ΔCT and PODXL-ΔDTHL) were
generated from pCMV6-PODXL-wt-DDK. Full-length human cortactin
(cortactin-wt) was obtained from Origene and subcloned into the pCMV6-green
fluorescent protein (GFP) vector. Cortactin-related mutants (cortactin-CT,
cortactin-NT, cortactin-SH3 and cortactin-ΔSH3) were generated from the
pCMV6-cortactin-GFP vector. All primers are listed in the Supplementary
Table S1, available at Carcinogenesis Online, and the reading frame sequence
Small-hairpin RNA vectors were obtained from the National RNAi Core
Facility (Academia Sinica, Taipei, Taiwan). To generate stable PODXL- and
cortactin-knockdown cell lines, HEK293T packaging cells were
cotransfected with the packaging plasmid (pCMV-∆R8.91), envelope (pMDG) and
hairpin pLKO-RNAi vectors using a PolyJET Transfection Kit (SignaGen
Laboratories, Ijamsville, MD). At 48 h post-transfection, virus-containing
supernatants were collected, mixed with fresh media containing polybrene
(8 μg/ml) and incubated with target cells for another 48 h. Transduced cells
were selected with puromycin (4 μg/ml) for 7 days.
For the orthotopic spontaneous metastasis assay, MDA-MB-231 (1 × 106) cells
were mixed with Matrigel (1:1) in a volume of 100 μl and were injected into
the fourth mammary fat pad of 6- to 8-week-old non-obese diabetic/severe
combined immunodeficiency (NOD/SCID) mice, which were then monitored
for 10–12 weeks. Primary tumor growth after an orthotopic injection was
measured using calipers every 3 days during the experiments and was calculated
using the following equation: length × (width)2 × 0.52. For the experimental
lung metastasis assay, tumor cells were injected into a tail vein of NOD/SCID
mice for 8 weeks. All dissected lungs were embedded with paraffin, and tissue
sections were stained with hematoxylin and eosin. Metastatic tumor colonies
were counted under a microscope. To examine macrometastases,
MDA-MB231-eGFP cells were orthotopically inoculated into NOD/SCID. Mice were
sacrificed at 12 weeks after inoculation, and tissues were excised and observed
under a fluorescence stereomicroscope to detect macrometastases. All animals
were cared for in a specific pathogen-free room and treated in accordance with
the animal care protocol approved by the Academia Sinica Animal Committee.
Rac1/Cdc42 activity was detected using a Rac1/Cdc42 activation assay kit
(Millipore, Bedford, MA), according to the manufacturer’s instructions. In
brief, cells were lysed with lysis buffer and directly incubated with PAK-1
PBD agarose at 4°C for 1 h on a rotator. Beads were washed three times with
wash buffer, resuspended in 5× Laemmli reducing sample buffer and
subjected to Western blotting. Wild-type (Addgene12975) and dominant negative
(T17N) Cdc42 (Addgene12601) plasmids were kindly provided by Dr Klaus
). The inhibitors of Rac1 and Cdc42 were obtained from Merck
(Merck KGaA, Darmstadt, Germany).
Total RNA was extracted using an RNeasy Plus Mini kit (Qiagen) and
reverse transcribed using SuperScript III reverse transcriptase (Invitrogen).
Quantitative PCR was performed using the resulting cDNA, LightCycler 480
SYBR Green I Master Mix (Roche, Basel, Switzerland) and a LightCycler 480
System (Roche). The results were expressed as fold change relative to the
control sample, using the equation ΔΔCT. GAPDHs were used as internal controls
for normalization. Primer sequences are listed in the Supplementary Table S2,
available at Carcinogenesis Online.
Western blotting and immunoprecipitation
Cells were lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate and
50 mM Tris-HCl at pH 7.4) containing protease and a phosphatase inhibitor
cocktail (Roche). For immunoprecipitation, cell lysates were precleared with
agarose-protein G for 1 h and incubated overnight at 4°C with the
appropriate protein G-conjugated primary antibodies. Beads were washed three times
with radioimmunoprecipitation assay buffer and were boiled in sample buffer
(50 mM Tris-HCl at pH 6.8, 2% sodium dodecylsulfate, 0.1% bromophenol
blue and 10% glycerol). Equal amounts of proteins were separated on sodium
dodecylsulfate–polyacrylamide gel electrophoresis and then transferred to
polyvinylidene difluoride membranes (Millipore). The membrane was blocked
with 1% bovine serum albumin/Tris Bufferred Saline Tween 20 and incubated
overnight with specific primary antibodies against PODXL (1:500, Santa Cruz
Biotechnology, Santa Cruz, CA), FAK (1:2000, Millipore), phospho-FAK
(1:2000, Millipore), paxillin (1:2000, Millipore), phospho-paxillin (1:500,
Millipore), DDK (1:5000, Origene), GFP (1:10 000, Origene), N-WASp
(1:1000, Millipore), cortactin (1:2000, Millipore), phospho-cortactin (1:2000,
Cell Signal Technology) or α-tubulin (1:5000, Sigma, St. Louis, MO).
Membranes were then incubated with the appropriate horseradish peroxidase–
conjugated secondary antibodies (1:50 000, Jackson ImmunoResearch) for 1 h
at room temperature, and proteins were detected using an enhanced
chemiluminescence kit (Millipore, Temecula, CA).
Transwell migration and invasion assays
Cells (105) were seeded in a transwell insert (8 μm filters, Corning) coated
with Matrigel (BD Biosciences, La Jolla, CA) or left uncoated for different
periods of time. After incubation, cells were fixed with 4% paraformaldehyde
for 10 min. The un-invaded cells were removed by a cotton swab; invaded cells
were stained with 4′,6-diamino-2-phenylindole, imaged under an inverted
fluorescent microscope (Zeiss). Data were derived from six random pictures.
Time-lapse migration assay
Cells at 2 × 104 were seeded on a Chamlide magnetic chamber with complete
growth media and were monitored using an inverted light microscope (Zeiss
HAL100 reflected-light microscope) under a temperature and CO control
tem. Images were captured every 10 min for 6 h. The distance of migration was
defined as the movement of the cell center per unit of time and was measured
using MetaMorph software (Universal Imaging, West Chester, PA).
Human colon and breast cancer tissue microarrays were purchased from
Biomax. The use of tissue microarrays was approved by the Human Subject
Research Ethics Committee, Institutional Review Board, Academia Sinica
(AS-IRB02-100067). Slides were deparaffinized, rehydrated and heated
in sodium citrate buffer (0.01 M, pH 6.0) for antigen retrieval. Slides were
immersed in 3% hydrogen peroxide in methanol to block endogenous
peroxidase activity and incubated with an anti-PODXL antibody (1:250, Atlas) for 1 h.
After washing three times with Phosphate Bufferred Saline Tween 20, a
detection reagent (Super Sensitive Polymer-HRP IHC Detection System, BioGenex)
and DAB chromogen were applied, followed by counterstaining with
hematoxylin. The tissue microarrays were scanned by TissueFAX (TissueGnostics,
Vienna, Austria), which were performed by two blind independent observers.
Invadopodia assay and gelatin degradation
To detect invadopodia, cells were stained with rhodamine-conjugated
phalloidin (1:350, Molecular Probe, Invitrogen) and a fluorescein
isothiocyanateconjugated anticortactin antibody (Millipore), before being observed under a
confocal microscope (TCS SP5; Leica, Wetzlar, Germany). To measure
invadopodia activity, cells were seeded onto Millicell EZ slides (Millipore) coated
with fluorescein isothiocyanateconjugated gelatin, according to the instructions
of the Gelatin Invadopodia Assay Kit (Millipore). After 16–48 h of incubation,
slides were fixed, permeabilized with Triton-X 100 (0.1% in
phosphate-buffered saline) and stained with rhodamine-conjugated phalloidin or cortactin.
Nuclei were counterstained with 4′,6-diamino-2-phenylindole (1:500). Slides
were examined and photographed under a confocal microscope. The activity
of invadopodia was defined by the average number of degradation puncta per
cell in at least eight random fields (
Gene expression data obtained from NCI-60 cancer cell lines using Affymetrix
HU-133A and U133B chips were downloaded from the CellMiner database
(http://discover.nci.nih.gov/cellminer/), and heat maps were drawn using
Microsoft Excel. P values are given for the medium-rank analysis.
All data were derived from at least three independent experiments. Values were
expressed as the mean ± standard error of the mean. Significant differences
were determined using two-tailed, unpaired Student’s t-test, unless otherwise
specified. P values of (*) <0.05 and (**) <0.01 were considered significant.
The correlation coefficient was assayed by a Pearson correlation.
Expression of PODXL is associated with breast cancer motility and
the metastatic capability
To evaluate the clinical relevance of PODXL to cancer malignancy,
we used immunohistochemistry analysis to examine PODXL protein
expression in 80 breast tumor tissues and their matched adjacent
normal counterparts. Results showed that the expression of PODXL was
significantly increased in tumor tissues (P < 0.001) (Figure 1A). We
further analyzed matched samples from primary and lymph node
metastatic breast tumors and detected an increased potential of PODXL
upregulation in lymph node metastatic tumor sites (n = 50, P < 0.001)
(Figure 1B), suggesting that increased expression of PODXL might
be associated with tumor metastasis.
Tumor metastasis encompasses several processes, such as
migration/invasion, extravasation and metastatic colonization. To
elucidate the effect of PODXL on tumor metastasis, we
investigated whether PODXL expression was correlated with the
migratory ability. We analyzed gene profiles from an NCI60 cancer
cell panel database to study the correlation between PODXL and
motility-related genes, such as E-cadherin (CDH1), CD44 and
vimentin. We found that PODXL expression was positively
correlated with CD44 (correlation coefficient = 0.425, P = 0.006)
and vimentin (correlation coefficient = 0.335, P = 0.03), both of
which are characteristic of basal-like and mobile cells (Figure 1C).
Conversely, CDH1, an adhesion and luminal-like cell marker,
was negatively correlated with PODXL (correlation coefficient =
−0.530, P = 0.001) (Figure 1C). A recent study also reported that
increased expression of PODXL is correlated with basal-like
phenotype of breast cancer (
). In agreement with previous study,
we found PODXL expression was positively correlated with CD44
and vimentin. In addition, a higher level of PODXL in basal-like
MDA-MB-231 cells exhibited a greater migratory capability than
did luminal-like MCF7 cells (Supplementary Figure S1A,
available at Carcinogenesis Online). Conversely, knockdown of PODXL
in MDA-MB-231 cells resulted in diminished cell migration and
invasion capabilities (Figure 1D and 1E). MDA/PODXL shRNA-1
and shRNA-2 showed similar effects on cell migration as that of
MDA/PODXL shRNA-3 cells (data not shown). Time-lapse
observations also revealed that suppression of PODXL significantly
decreased cell migratory rate (Figure 1F). Conversely,
overexpression of PODXL in MCF7 cells increased cell migration and
invasion (Supplementary Figure S1B, available at Carcinogenesis
Online), suggesting that expression of PODXL is crucial for tumor
PODXL regulates invadopodia formation
We observed that suppression of PODXL impacted cell morphology
in MDA-MB-231 cells. The morphology of mock cells displayed
a spindle-like appearance, whereas the morphology of
PODXLknockdown cells showed irregular characteristics and a rounded
and swelling appearance (Figure 2A, left panel). Quantitative data
showed that suppression of PODXL drastically increased cell area,
consistent with our findings. These results suggest that PODXL
might play a role in actin architecture (Figure 2A, upper right).
We further stained F-actin, and the results showed that a dynamic
lamellar structure was observed in mock cells but was lost in
PODXL-knockdown cells (Supplementary Figure S2A, available at
Carcinogenesis Online). Actin-enriched lamellipodia, which refer
to migratory cells, were observed at the leading edge of lamella
in mock cells (Figure 2A, middle and right panels). The
percentage of cells with lamellipodia among PODXL-knockdown cells
decreased from 12.6 to 3.1%, compared with that of mock cells.
In addition, knockdown of PODXL resulted in loss of actin
polarity and directionality (Figure 2A, lower panel). Given that FAK
and paxillin are crucial for lamellipodia formation, we examined
the effects of PODXL on FAK and paxillin. The results showed
that knockdown of PODXL decreased phosphorylation of FAK and
paxillin (Figure 2B, left panel), whereas the phosphorylation of
FAK and paxillin was increased in PODXL-overexpressing cells
(Supplementary Figure S2B), available at Carcinogenesis Online,
suggesting that the induction of FAK/paxillin activation may be
involved in PODXL-mediated lamellipodia formation. Because
the formation of invadopodia plays an important role in the
degradation of local ECM regions, which facilitates tumor invasion,
we wondered whether PODXL affected invadopodia formation.
MDA-MB-231 cells were co-stained with F-actin and cortactin,
which are often used as indicators of invadopodia precursors. In
mock cells, we found that most of the F-actin and cortactin were
colocalized at the cell periphery, implying nascent focal adhesion in
migratory cells (Figure 2C, arrow indicated). We also observed that
some of the F-actin/cortactin colocalizations formed a
punctatelike structure in the cytoplasm, implying an invadopodia precursor
(Figure 2C, triangle indicated). However, in PODXL-knockdown
cells, colocalization of F-actin/cortactin was lost at both the
membrane edge and in the cytoplasm (Figure 2C). Moreover, the
phosphorylation of cortactin and N-WASp, two important mediators in
invadopodia formation, was decreased in PODXL-knockdown cells
(Figure 2B, right panel). To further verify that PODXL can regulate
invadopodia function, we used fluorescein
isothiocyanate–conjugated gelatin degradation assay to monitor invadopodial activity. As
shown in Figure 2D, accumulation of cortactin in the perinucleus
and membrane edge were found to be colocalized with the gelatin
degradation puncta in the mock cells. The z-stack projection images
showed the invasion of cortactin into the gelatin layer, implying
invadopodial protrusions. However, the colocalization of cortactin
with gelatin degradation puncta was blocked in MDA/shPODXL
cells (Figure 2D). Conversely, overexpression of PODXL in MCF7
cells induced colocalization of F-actin and cortactin in both the cell
periphery and cytoplasm, compared with mock cells (Figure 2E).
PODXL overexpression promoted the invasion of F-actin and
cortactin into the gelatin layer (Supplementary Figure S3, available at
Carcinogenesis Online) and increased gelatin degradation as well
(Figure 2F). These data indicate that PODXL expression plays a
crucial role in invadopodia formation and activation.
Suppression of PODXL downregulates cortactin-mediated
Expression of cortactin is crucial for invadopodia formation and
activation through assembling actin-nucleating molecules. To further
confirm the importance of PODXL in invadopodia-mediated tumor
invasiveness, we compared PODXL-knockdown alone with
cortactinknockdown cells. Results showed that suppression of cortactin
significantly inhibited tumor invasion (Supplementary Figure S4, available
at Carcinogenesis Online). In addition, suppression of PODXL alone
significantly diminished invadopodial activity, similar to that in
cortactin-knockdown cells (Figure 3A). Moreover, suppression of both
PODXL and cortactin markedly abrogated invadopodial activity
(Figure 3A) and the invasive ability (Figure 3B). However, inhibition
of gelatin degradation in PODXL-knockdown cells was rescued when
cortactin was reintroduced (Figure 3C). We also observed that the
reintroduction of cortactin (AS2_Cortactin) into PODXL-knockdown
cells restored cytoskeleton polarity (data not shown). These data
suggest that cortactin participates in PODXL-mediated invadopodia
formation and activation.
Rac1/Cdc42 participates in PODXL-mediated cortactin activation
We further investigated the molecular mechanism by which PODXL
induces invadopodia formation. Immunoprecipitation-Western
blot analysis revealed that PODXL was associated with cortactin
(Figure 4A). Immunofluorescence analysis showed that some of
the cortactin colocalized with PODXL in the plasma membrane
(Figure 4B). These findings were confirmed by
co-immunoprecipitation of DDK-tagged PODXL with cortactin-GFP (Figure 4C).
Previous studies showed that the DTHL residue in the C-terminal
tail of PODXL is required for binding of the Ezrin/Na+/H+ exchanger
regulatory factor complex, which supports actin reorganization
). As such, we tested whether the DTHL motif of PODXL is
required for the association with cortactin. Co-immunoprecipitation
experiments showed that deletion of the DTHL motif of PODXL
(PODXL-ΔDTHL) resulted in the loss of binding between the
protein and cortactin (Figure 4D). Similar results were found after
deletion of the intracellular domain (PODXL-ΔCT) of PODXL
(Figure 4D), suggesting that the DTHL residue participates in the
association with cortactin. To further map the region of cortactin
required for association with PODXL, a series of truncation mutants
was generated (Figure 4E). Co-immunoprecipitation experiments
revealed that PODXL associated with the N-terminal domain, but
not the SH3 or the C-terminal domain of cortactin (Figure 4E).
In addition, overexpression of wild-type PODXL (PODXL-wt)
increased cortactin phosphorylation (Figure 4F) and invadopodia
activation as well (Figure 4G); however, these activations were
reduced in both PODXL-ΔDTHL and PODXL-ΔCT transfectants
(Figure 4F and G).
Because a previous study reported that the DTHL motif in PODXL
is required for Rac1 activation (
), and Rac1 and Cdc42 can regulate
cortactin activation, we next examined whether Rac1 and/or Cdc42
participated in PODXL-mediated activating signaling in invadopodia.
Results showed that knockdown of PODXL in MDA-MB-231 cells
abrogated Rac1 and Cdc42 activity (Figure 5A). Similar results were
found in cortactin-knockdown and PODXL/cortactin-knockdown
cells (Figure 5A). In addition, we found that PODXL induced Cdc42
activity, whereas it was diminished in cells transfected with
PODXLΔDTHL or PODXL-ΔCT (Figure 5B). Moreover, overexpression of
the dominant-negative Cdc42 (T17N) impeded PODXL-elicited
cortactin phosphorylation (Figure 5C). Inhibition of Rac1 or Cdc42 by
specific chemical inhibitors abrogated cortactin phosphorylation and
reduced the association of cortactin with PODXL (Figure 5D). These
data suggest that the DTHL motif in PODXL might be important in
triggering Rac1/Cdc42/cortactin-mediated invadopodia activation.
PODXL induces activation of FAK and paxillin through Rac1/
The aforementioned data identified that suppression of PODXL
downregulated FAK and paxillin phosphorylation (Figure 2B), and both
are critical for lamellipodial induction. It is still unclear how PODXL
regulates FAK and paxillin. 293T cells were transfected with different
mutants of PODXL, and results showed that PODXL-wt induced
phosphorylation of FAK and paxillin (Figure 5E). However, phosphorylation
of FAK and paxillin decreased in cells transfected with PODXL-ΔDTHL
or PODXL-ΔCT (Figure 5E). Moreover, inhibition of Rac1 and Cdc42
in 293T/PODXL cells effectively suppressed FAK and paxillin
phosphorylation (Figure 5F). Treatment of cells with Src inhibitor (PP2)
blocked phosphorylation of FAK and paxillin. Similar results were
observed in cells treated with the Src-specific inhibitor (SU6656), which
has high affinity and selectivity for Src kinase, relative to PP2 which are
also inhibitors of PDGFR and FAK (Supplementary Figure S5, available
at Carcinogenesis Online). These data indicate that Src may participate
in PODXL-induced FAK and paxillin activation, and that activation of
Rac1/Cdc42 by PODXL is crucial for FAK and paxillin activation.
Inhibition of PODXL suppresses breast tumor metastasis
To demonstrate that PODXL indeed contributes to tumor metastasis,
MDA-MB-231/shPODXL cells were intravenously injected into tail
vein of NOD/SCID mice. Results showed that knockdown of PODXL
effectively suppressed tumor colonization and nodules formation in
mice lungs (Figure 6A). Moreover, MDA-MB-231-eGFP cells were
orthotopically inoculated into NOD/SCICD mice for 12 weeks, and
systemic metastases were observed. In the control group, six of seven
mice contained metastatic tumors in the lungs, and four of seven mice
had macrometastases in the brain, liver, or spleen. However,
suppression of PODXL significantly inhibited tumor dissemination to both the
lungs and other tissues (Figure 6B). No preferential organ for metastasis
was observed in PODXL-knockdown cells (Figure 6B). Knockdown of
PODXL significantly impeded tumor dissemination but had modest
inhibition on primary tumor volumes (Supplementary Figure S6A, available
at Carcinogenesis Online). We did not observe significant growth
inhibition in PODXL knockdown cells in the in vitro culture condition
according to the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide
assay (data not shown), suggesting that other factors might regulate tumor
growth in the tumor microenvironment. Analysis of mRNA level in the
primary tumor tissues showed that the pro-metastatic cytokines and
chemokines, including IL1β, IL8, IL24, LOX and LOXL4, were
downregulated in PODXL knockdown tumor tissues (Supplementary Figure
S6B, available at Carcinogenesis Online). Several studies have reported
that these molecules act as chemoattractants to support tumor growth in
the tumor microenvironment, and that promotes tumor progression. These
data indicate that PODXL participates in not only promotion of
invadopodia activity but also regulation of metastasis gene expression. Moreover,
suppression of PODXL showed a similar inhibitory effect as
cortactinknockdown on tumor metastasis (Figure 6C). Taken together, these results
emphasize that PODXL expression is critical for tumor metastasis.
Previous studies have reported that overexpression of PODXL is
associated with poor prognoses features. In breast cancer, high
expression of PODXL is correlated with lymphatic invasion. However, it is
still unclear whether PODXL participates in tumor metastasis, and
the role of PODXL and its underlying mechanism are still unknown.
In the current study, we found that increased expression of PODXL
was detected in lymph node metastatic breast cancer cells. We
further provided evidence showing that PODXL promotes invadopodia
activity through Rac1/Cdc42-mediated FAK and cortactin signaling.
Knockdown of PODXL significantly suppressed tumor
dissemination. These data provide direct evidence demonstrating that PODXL
plays an important role in tumor metastases.
PODXL is located on chromosome 7q32-q33, a region that is
frequently associated with allelic imbalances and aggressive prostate
). In addition to prostate cancer, upregulation of PODXL
is associated with poor prognoses of breast, colorectal and
ovarian cancers (
). The molecular mechanism underlying the role
of PODXL to the contribution of tumor progression is still unclear.
We found that PODXL expression was closely correlated with the
mesenchymal cell markers, CD44 and vimentin (Figure 1C), which
are frequently associated with cell motility. Suppression of PODXL
inhibited tumor migratory and invasive capabilities (Figure 1E and
F), and restricted tumor dissemination (Figure 6A–C). Clinical
analyses also revealed that expression of PODXL was increased in lymph
node metastatic cancers (Figure 1B). These data strongly indicate that
PODXL expression contributes to tumor metastasis.
The formation of invadopodia endows cells with the ability to
erode local ECM regions via trafficking matrix metalloproteinases to
the tips of invadopodia (
). Cortactin plays a central role in the
formation of lamellipodia and invadopodia through gathering of the
actin nucleation factors, Arp2/3 and N-WASp, which further enhances
elongation of branch actin (
). Cortactin acts as a bridge to
cluster actin filament and N-WASp. N-WASp is responsible for
carrying out actin nucleation through interaction with Arp2/3. The
activation of cortactin and N-WASp enhances actin polymerization, and the
colocalization of cortactin and F-actin promotes filopodia and
invadopodia formation. Colocalization of cortactin with F-actin at the cell
periphery is referred to as nascent focal adhesion (
), whereas their
colocalization in the cytoplasm indicates the formation of
invadopodia precursors (
). Cdc42 and Rac1 play important roles in cortactin
activation, cortactin-mediated actin polymerization and the formation
of lamellipodia (
). They also enhance actin polymerization by
promoting the binding of N-WASp to F-actin (
). Activation of
Rac1 has been reported to be involved in the PODXL-mediated
antiadhesive characteristic (23). The roles of Cdc42/Rac1 and cortactin in
PODXL-induced cell invasiveness, however, are still unclear.
We found that PODXL expression was crucial for actin
reorganization. Suppression of PODXL significantly induced cell
morphological and behavior changes (Figure 2A). PODXL knockdown
MDA-MB-231 cells showed irregular and rounded appearance,
compared with mock cells, which showed spindle-liked cell shape. Rac1/
Cdc42, cortactin and N-WASp are important molecules involved in
the nucleation of actin polymerization. In addition, FAK and ERM
proteins, including Ezrin, participate in the linkage of actin filaments
to the plasma membrane. The formation of actin filaments maintains
cell shape, structure and locomotion; however, these molecules are
suppressed by PODXL silencing, suggesting that PODXL is crucial
for cytoskeleton maintenance.
Reciprocal activation of Rac1/Cdc42 and FAK was reported to
be involved in cell spread and extracellular signal-regulated kinase
). In addition, FAK phosphorylates cortactin at
tyrosines Y421, Y466 and Y482 (42), and the association of cortactin
with the FAK/Src complex regulates cell motility (
of Src also plays an important role in modulating cortactin
phosphorylation and invadopodia formation. We found that Cdc42/Rac1 is
important to PODXL-induced cortactin phosphorylation. Inhibition
of Cdc42/Rac1 and Src downregulates PODXL-induced FAK and
paxillin phosphorylation. These data support the notion that PODXL
activates Rac1/Cdc42/cortactin and the FAK signaling network,
leading to promotion of lamellipodia and invadopodia formation.
Previous studies demonstrated that the association of Ezrin with
PODXL regulates tumor mobility. In addition, activation of Rac1 by
PODXL via binding of the Rac1 guanine exchange factor, ARHGEF7,
contributes to cell aggressiveness (
). Rac1 can trigger cortactin
translocation to the cell periphery (
), probably through PAK1,
downstream of Rac1/Cdc42. Activation of Rac1/Cdc42 leads to triggering
of the reorganization of cortactin in the lamella spreading site and
its association with N-WASp. The mechanism underlying
PODXLmediated invadopodia formation, however, is still unclear. We found
that the DTHL motif in PODXL is crucial for Rac1/Cdc42
activation and subsequent phosphorylation of cortactin. The N-terminal
domain of cortactin is required for binding of Arp2/3 and subsequent
F-actin nucleation (
), whereas phosphorylation at tyrosine 421 in the
C-terminal domain is involved in the Rac1-mediated cortical actin
). Rac1/Cdc42 was reported to participate in
cortactinmediated lamella spreading in platelet cells (
). We propose that
activation of Rac1/Cdc42 is required for PODXL to induce the
phosphorylation and translocation of cortactin to cell membranes. Because
activation of cortactin regulates Rac1/Cdc42 activity, the reciprocal
activation of cortactin and Rac1/Cdc42 by PODXL enhances
lamellipodia and invadopodia formation. However, it is worthwhile
deciphering the direct binding region between PODXL and cortactin with
in vitro binding assay.
Our data are the first to report that PODXL regulates
invadopodia through Rac1/Cdc42-mediated cortactin activation. PODXL is
important to cell adhesion, migration and invasion by cooperating
with actin reorganization factors. Our unpublished data also found that
PODXL regulates genes involved in ECM degradation, wound healing
and the epithelial–mesenchymal transition, which are characteristics
of tumor metastases. However, the detailed regulatory mechanism still
needs to be investigated. Taken together, we identified that
overexpression of PODXL is strongly associated with tumor malignancy
progression, it could be a potential biomarker for cancer diagnosis, and as such,
PODXL might be an attractive therapeutic target for cancer treatment.
Supplementary Tables S1 and S2 and Figures S1–S6 can be found at
Academia Sinica and the National Science Council
(NSC1012321-B-001-021 and NSC101-2325-B-001-010 to H.C.W. and
NSC102-2320-B-038-005 to C.W.L.).
We thank the Core Facility of the Institute of Cellular and Organismic Biology
and the RNAi Core, Academia Sinica for technical support.
Conflict of Interest Statement: None declared.
1. Poincloux , R. et al. ( 2009 ) Matrix invasion by tumour cells: a focus on MT1-MMP trafficking to invadopodia . J. Cell Sci., 122(Pt 17) , 3015 - 3024 .
2. Yamaguchi ,H. et al. ( 2009 ) Lipid rafts and caveolin-1 are required for invadopodia formation and extracellular matrix degradation by human breast cancer cells . Cancer Res. , 69 , 8594 - 8602 .
3. Artym , V.V. et al. ( 2006 ) Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function . Cancer Res. , 66 , 3034 - 3043 .
4. Chan ,K.T. et al. ( 2009 ) FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion . J. Cell Biol ., 185 , 357 - 370 .
5. Murphy , D.A. et al. ( 2011 ) The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function . Nat. Rev. Mol. Cell Biol ., 12 , 413 - 426 .
6. Oser , M. et al. ( 2009 ) Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation . J. Cell Biol ., 186 , 571 - 587 .
7. Weed , S.A. et al. ( 2000 ) Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex . J. Cell Biol ., 151 , 29 - 40 .
8. Lai , F.P. et al. ( 2008 ) Arp2/3 complex interactions and actin network turnover in lamellipodia . EMBO J ., 27 , 982 - 992 .
9. Bryce , N.S. et al. ( 2005 ) Cortactin promotes cell motility by enhancing lamellipodial persistence . Curr. Biol ., 15 , 1276 - 1285 .
10. Sung , B.H. et al. ( 2011 ) Cortactin controls cell motility and lamellipodial dynamics by regulating ECM secretion . Curr. Biol ., 21 , 1460 - 1469 .
11. Eckert , M. A . et al. ( 2011 ) Twist1-induced invadopodia formation promotes tumor metastasis . Cancer Cell , 19 , 372 - 386 .
12. Li , X. et al. ( 2008 ) Aberrant expression of cortactin and fascin are effective markers for pathogenesis, invasion, metastasis and prognosis of gastric carcinomas . Int. J. Oncol ., 33 , 69 - 79 .
13. Clark , E.S. et al. ( 2007 ) Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia . Cancer Res. , 67 , 4227 - 4235 .
14. Kerjaschki , D. et al. ( 1984 ) Identification and characterization of podocalyxin-the major sialoprotein of the renal glomerular epithelial cell . J. Cell Biol ., 98 , 1591 - 1596 .
15. Kerjaschki , D. et al. ( 1986 ) Identification of a major sialoprotein in the glycocalyx of human visceral glomerular epithelial cells . J. Clin. Invest ., 78 , 1142 - 1149 .
16. Sassetti , C. et al. ( 2000 ) Identification of endoglycan, a member of the CD34/podocalyxin family of sialomucins . J. Biol. Chem ., 275 , 9001 - 9010 .
17. Fieger , C. B . et al. ( 2003 ) Endoglycan, a member of the CD34 family, functions as an L-selectin ligand through modification with tyrosine sulfation and sialyl Lewis x . J. Biol. Chem ., 278 , 27390 - 27398 .
18. Doyonnas , R. et al. ( 2005 ) Podocalyxin is a CD34-related marker of murine hematopoietic stem cells and embryonic erythroid cells . Blood , 105 , 4170 - 4178 .
19. Choo , A.B. et al. ( 2008 ) Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1 . Stem Cells , 26 , 1454 - 1463 .
20. Horvat , R. et al. ( 1986 ) Endothelial cell membranes contain podocalyxinthe major sialoprotein of visceral glomerular epithelial cells . J. Cell Biol ., 102 , 484 - 491 .
21. Takeda , T. et al. ( 2001 ) Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton . J. Clin. Invest ., 108 , 289 - 301 .
22. Takeda , T. et al. ( 2000 ) Expression of podocalyxin inhibits cell-cell adhesion and modifies junctional properties in Madin-Darby canine kidney cells . Mol. Biol. Cell , 11 , 3219 - 3232 .
23. Hsu , Y.H. et al. ( 2010 ) Podocalyxin EBP50 ezrin molecular complex enhances the metastatic potential of renal cell carcinoma through recruiting Rac1 guanine nucleotide exchange factor ARHGEF7 . Am. J. Pathol ., 176 , 3050 - 3061 .
24. Sizemore , S. et al. ( 2007 ) Podocalyxin increases the aggressive phenotype of breast and prostate cancer cells in vitro through its interaction with ezrin . Cancer Res. , 67 , 6183 - 6191 .
25. Somasiri , A. et al. ( 2004 ) Overexpression of the anti-adhesin podocalyxin is an independent predictor of breast cancer progression . Cancer Res. , 64 , 5068 - 5073 .
26. Larsson , A. et al. ( 2011 ) Overexpression of podocalyxin-like protein is an independent factor of poor prognosis in colorectal cancer . Br. J. Cancer , 105 , 666 - 672 .
27. Cipollone , J.A. et al. ( 2012 ) The anti-adhesive mucin podocalyxin may help initiate the transperitoneal metastasis of high grade serous ovarian carcinoma . Clin. Exp. Metastasis , 29 , 239 - 252 .
28. Forse , C.L. et al. ( 2013 ) Elevated expression of podocalyxin is associated with lymphatic invasion, basal-like phenotype, and clinical outcome in axillary lymph node-negative breast cancer . Breast Cancer Res. Treat. , 137 , 709 - 719 .
29. Nalbant , P. et al. ( 2004 ) Activation of endogenous Cdc42 visualized in living cells . Science , 305 , 1615 - 1619 .
30. Smith-Pearson , P.S. et al. ( 2010 ) Abl kinases are required for invadopodia formation and chemokine-induced invasion . J. Biol. Chem ., 285 , 40201 - 40211 .
31. Schmieder , S. et al. ( 2004 ) Podocalyxin activates RhoA and induces actin reorganization through NHERF1 and Ezrin in MDCK cells . J. Am. Soc. Nephrol. , 15 , 2289 - 2298 .
32. Casey , G. et al. ( 2006 ) Podocalyxin variants and risk of prostate cancer and tumor aggressiveness . Hum. Mol. Genet ., 15 , 735 - 741 .
33. Kempiak , S.J. et al. ( 2005 ) A neural Wiskott-Aldrich Syndrome proteinmediated pathway for localized activation of actin polymerization that is regulated by cortactin . J. Biol. Chem ., 280 , 5836 - 5842 .
34. MacGrath , S.M. et al. ( 2012 ) Cortactin in cell migration and cancer at a glance . J. Cell Sci., 125(Pt 7) , 1621 - 1626 .
35. Vidal , C. et al. ( 2002 ) Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin . Blood , 100 , 4462 - 4469 .
36. Lai , F.P. et al. ( 2009 ) Cortactin promotes migration and platelet-derived growth factor-induced actin reorganization by signaling to Rho-GTPases . Mol. Biol. Cell , 20 , 3209 - 3223 .
37. Hüfner , K. et al. ( 2002 ) The acidic regions of WASp and N-WASP can synergize with CDC42Hs and Rac1 to induce filopodia and lamellipodia . FEBS Lett ., 514 , 168 - 174 .
38. Rohatgi , R. et al. ( 2000 ) Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate . J. Cell Biol ., 150 , 1299 - 1310 .
39. Prehoda , K.E. et al. ( 2000 ) Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex . Science, 290 , 801 - 806 .
40. Flinder , L.I. et al. ( 2011 ) EGF-induced ERK-activation downstream of FAK requires rac1-NADPH oxidase . J. Cell. Physiol. , 226 , 2267 - 2278 .
41. Chang , F. et al. ( 2011 ) Tyrosine phosphorylation of Rac1: a role in regulation of cell spreading . PLoS One , 6 , e28587 .
42. Tomar , A. et al. ( 2012 ) Cortactin as a target for FAK in the regulation of focal adhesion dynamics . PLoS One , 7 , e44041 .
43. Wang , W. et al. ( 2011 ) Tyrosine phosphorylation of cortactin by the FAKSrc complex at focal adhesions regulates cell motility . BMC Cell Biol ., 12 , 49 .
44. Wu , X. et al. ( 2004 ) Focal adhesion kinase regulation of N-WASP subcellular localization and function . J. Biol. Chem ., 279 , 9565 - 9576 .
45. Weed , S.A. et al. ( 1998 ) Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1 . J. Cell Sci., 111(Pt 16) , 2433 - 2443 .
46. Head , J.A. et al. ( 2003 ) Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton . Mol. Biol. Cell , 14 , 3216 - 3229 .