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Ligand-directed targeting of lymphatic vessels
uncovers mechanistic insights in melanoma metastasis
Dawn R. Christianson
a,1,2
, Andrey S. Dobroff
b,c,1
, Bettina Proneth
a,1,3
, Amado J. Zurita
d
, Ahmad Salameh
a,4
,
Eleonora Dondossola
a
, Jun Makino
e
, Cristian G. Bologa
f
, Tracey L. Smith
b,c
, Virginia J. Yao
b,c
, Tiffany L. Calderone
g
,
David J. O’Connell
h
, Tudor I. Oprea
f
, Kazunori Kataoka
i
, Dolores J. Cahill
h
, Jeffrey E. Gershenwald
g
, Richard L. Sidman
j,5
,
Wadih Arap
b,k,5
, and Renata Pasqualini
b,c,5
a
David H. Koch Center and Departments of
d
Genitourinary Medical Oncology and
g
Surgical Oncology, The University of Texas MD Anderson Cancer Center,
Houston, TX 77030;
b
University of New Mexico Cancer Center and Divisions of
c
Molecular Medicine,
f
Translational Informatics, and
k
Hematology and
Medical Oncology, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131;
e
Center for Disease Biology
and Integrative Medicine, Graduate School of Medicine, and
i
Department of Bioengineering, Graduate School of Engineering, University of Tokyo, Tokyo
113-0033, Japan;
h
Conway Institute of Biomedical and Biomolecular Science, University College Dublin, Belfield, Dublin 4, Ireland; and
j
Department of
Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
Contributed by Richard L. Sidman, January 9, 2015 (sent for review December 5, 2014)
Metastasis is the most lethal step of cancer progression in patients
with invasive melanoma. In most human cancers, including mela-
noma, tumor dissemination through the lymphatic vasculature
provides a major route for tumor metastasis. Unfortunately, molec-
ular mechanisms that facilitate interactions between melanoma cells
and lymphatic vessels are unknown. Here, we developed an un-
biased approach based on molecular mimicry to identify specific
receptors that mediate lymphatic endothelial–melanoma cell interac-
tions and metastasis. By screening combinatorial peptide libraries
directly on afferent lymphatic vessels resected from melanoma
patients during sentinel lymphatic mapping and lymph node biop-
sies, we identified a significant cohort of melanoma and lymphatic
surface binding peptide sequences. The screening approach was
designed so that lymphatic endothelium binding peptides mimic cell
surface proteins on tumor cells. Therefore, relevant metastasis and
lymphatic markers were biochemically identified, and a comprehen-
sive molecular profile of the lymphatic endothelium during mela-
noma metastasis was generated. Our results identified expression
of the phosphatase 2 regulatory subunit A, α-isoform (PPP2R1A)
on the cell surfaces of both melanoma cells and lymphatic endothe-
lial cells. Validation experiments showed that PPP2R1A is expressed
on the cell surfaces of both melanoma and lymphatic endothelial
cells in vitro as well as independent melanoma patient samples.
More importantly, PPP2R1A-PPP2R1A homodimers occur at the cellu-
lar level to mediate cell–cell interactions at the lymphatic–tumor in-
terface. Our results revealed that PPP2R1A is a new biomarker for
melanoma metastasis and show, for the first time to our knowledge,
an active interaction between the lymphatic vasculature and mela-
noma cells during tumor progression.
cell–cell interaction
|
cell surface peptide
|
lymphatic targeting
|
phage display
Despite the similarities between the lymphatic and blood
vasculature, lymphatic vessels (LyVs) have their own dis-
tinct morphological and molecular profile (1, 2). The lymphatic
vasculature plays a critical role in the pathogenesis of many
diseases, including inflammatory disorders, lymphedema, tumor
progression, and metastasis. The role of LyVs in tumor dissem-
ination and metastasis has been recognized and correlates with
the number of tumor-associated LyVs with lymph node (LN)
metastasis (3–5). In fact, the degree of LN involvement has be-
come a good prognostic indicator of patient outcome in several
human tumors, including melanoma (6). Surgical resection of the
primary tumor and regional LN is the best treatment option for
early-stage disease. However, early detection of in-transit mela-
noma or successful identification of metastasis-positive LN remains
a major challenge for clinicians because of the lack of reliable LN
markers and sensitive detection techniques.
As the malignant phenotype of melanoma cells transitions
from the noninvasive radial growth phase to the vertical growth
phase (metastatic phenotype), the repertoire of proteins expressed
on the cell surface, such as adhesion molecules and matrix-
degrading enzymes, does as well (7–9). These molecular changes
enable complex interactions of metastatic cells with the extracel-
lular milieu to propel the spread of disease. Moreover, growing
evidence implicates that soluble tumor factors, such as VEGF-C
and VEGF-D, induce lymphangiogenesis in LNs before the arrival
of tumor cells, thus creating a so-called “premetastatic niche”(10–
13). However, the mechanisms by which tumor cells leave the
primary tumor, invade the lymphatic system, and spread to re-
gional LN and distant organs are highly complex and may involve
additional yet to be identified molecules that facilitate cell–cell
attraction, adhesion, and migration. Here, we developed a rapid
ex vivo screening technology based on molecular mimicry to
identify novel cell–cell interacting ligands involved in the interplay
between tumor cells and lymphatic endothelial cells (LECs) that
potentially promote metastasis.
Significance
Formation of metastasis is the most deadly step in melanoma
progression and primarily occurs through the lymphatic vas-
culature. Unfortunately, little is known regarding the underlying
molecular mechanisms that enable interactions between mela-
noma cells and lymphatic vessels. In this study, we developed an
unbiased approach using combinatorial peptide libraries dis-
played on phage coat proteins to identify cell–cell interacting
proteins at the lymphatic vessel–tumor cell interface. Successful
application of this approach led to the identification of cell sur-
face PPP2R1A on both melanoma and lymphatic cell surfaces,
and more importantly, PPP2R1A facilitates tumor cell–lymphatic
endothelial cell interactions during melanoma cell metastasis.
Author contributions: D.R.C., A.S.D., B.P., A.J.Z., E.D., D.J.C., J.E.G., R.L.S., W.A., and R.P.
designed research; D.R.C., A.S.D., B.P., A.J.Z., A.S., E.D., J.M., T.L.C., and D.J.O. performed
research; D.R.C., A.S.D., B.P., A.J.Z., E.D., J.M., C.G.B., and T.I.O. analyzed data; and D.R.C.,
A.S.D., B.P., E.D., T.L.S., V.J.Y., K.K., J.E.G., R.L.S., W.A., and R.P. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1
D.R.C., A.S.D., and B.P. contributed equally to this work.
2
Present address: Arrowhead Research Corporation, Pasadena, CA 91101.
3
Present address: Helmholtz Zentrum München, Neuherberg 85764, Germany.
4
Present address: Brown Foundation Institute of Molecular Medicine, University of Texas
Health Science Center at Houston, Houston, TX 77030.
5
To whom correspondence may be addressed. Email: richard_sidman@hms.harvard.edu,
warap@salud.unm.edu, or rpasqual@salud.unm.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1424994112/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1424994112 PNAS
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Phage display has been successfully exploited in vitro, ex vivo,
and in vivo to decipher the molecular diversity of peptide binding
specificity to isolated proteins, purified antibodies (Abs), cell
surface receptors, and intracellular/cytoplasmic domains (14–
17). The novelty of our lymphatic screen was the availability of
intact human afferent LyVs obtained from melanoma patients
during intraoperative lymphatic mapping and sentinel LN bi-
opsies (18, 19). Additional studies on selected peptides allowed
us to biochemically identify several relevant metastasis and
lymphatic markers to generate a comprehensive molecular pro-
file of the lymphatic endothelium during melanoma metastasis.
We identified cell surface expression of phosphatase 2 regulatory
subunit A, α-isoform (PPP2R1A) (20) on both melanoma cells
and LECs. Furthermore, in vitro studies revealed that PPP2R1A
mediates a heterotypic cell–cell interaction through homodimer
formation. Finally, PPP2R1A is expressed at high levels in mela-
noma patient samples, thus highlighting its functional role during
melanoma progression and metastasis.
Results
Mapping the Lymphatic Endothelium in Melanoma. To map specific
proteins that participate in LEC–melanoma cell interactions, we
developed a unique two-arm screening method based on phage
display (step 1) (Fig. 1A) and the “molecular mimicry”approach
(step 2) (Fig. 1B). In step 1, segments of afferent LyVs were
isolated from residual tissues of sentinel LN biopsy procedures
from melanoma patients. A combinatorial peptide library dis-
played on phage, comprising seven random amino acid peptides
constrained by two cysteines (CX
7
C), was screened directly in
the lumen of isolated LyV. In each consecutive round, the
enriched phage pool from the previous round was used as the
input. In round 1, LyV segments of six patients were used to
account for interpatient variability and ensure an adequately
diverse phage pool for round 2 on LyV and primary LECs. For
rounds 2 and 3, LyV segments from four different patients were
used (n=14 patient-derived LyVs). In parallel, the peptides
enriched from round 1 from the LyV screening were subjected to
three additional rounds of screening with early-passage LECs.
LEC screenings (rounds 2–4) were carried out with the Bio-
panning and Rapid Analysis of Selective Interactive Ligands
approach (21, 22). Phages recovered from each round, except
round 1, were sequenced, and peptide abundance was de-
termined. Four peptides (GLTFKSL, VSQRNEL, FSGWSTV,
and AEKSSYV) were enriched (Fig. 1C). These enriched sequen-
ces were also present in the phage pool recovered after round 3 of
the LyV screening, indicating that the identified peptides bind to a
receptor accessible from the luminal side.
Next (step 2), rabbits were immunized against the four enriched
peptides, and polyclonal Abs were affinity-purified. To test if the
antipeptide sera recognized cell surface proteins on melanoma
cells, immunofluorescence experiments were performed with the
sera as a primary Ab pool. Anti-GLTFKSL Abs bound strongly to
a protein expressed on the cell surface in a high percentage of
C8161 melanoma cells (Fig. 1D), and very little binding was found
with the AEKSSYV, FSGWSTV, or VSQRNEL Abs. C8161
cells were not immunoreactive against the preimmune control
serum. Based on these results, we decided to pursue further
studies with the GLTFKSL ligand/receptor pair. To validate
additionally that the GLTFKSL peptide mimics a relevant
membrane protein, we performed a phage-based binding assay
with LECs derived from melanoma patients, human umbilical
vein endothelial cells, and C8161 melanoma cells. Phage dis-
playing the GLTFKSL peptide preferentially bound to LECs as
well as C8161 cells compared with human umbilical vein endo-
thelial cells, whereas an insertless control phage (Fd phage) did not
bind any of the cell types (Fig. 1E). Immunofluorescence studies
were performed on human dermal LECs (HDLECs) or LECs with
intact membranes (nonpermeabilized) and permeabilized cells to
confirm the specificity of the anti-GLTFKSL Ab to both lymphatic
and melanoma cells (Fig. 1F). In permeabilized cells, the
GLTFKSL Ab specifically stained the intracellular compartment.
In contrast, when cells with intact membranes were used, a distinct
cell surface immunostaining was observed for both LECs and
melanoma cells. Collectively, these results indicated that the
GLTFKSL ligand/receptor pair is involved in lymphatic endothe-
lium–melanoma cell interactions.
To identify the endogenous protein that the GLTFKSL pep-
tide mimics, anti-GLTFKSL serum was exposed to a human
Fig. 1. Two-step screening methodology based on molecular mimicry.
(A) Flowchart depicting the separate screenings based on an LyV culture and
consecutive LEC screening rounds with the Biopanning and Rapid Analysis of
Selective Interactive Ligands (BRASIL) method. (B) Identification of the na-
tive protein-mimicking ligands with high-density protein microarrays. Anti-
peptide serum and control serum reactivity to proteins on a nitrocellulose-
dotted human microarray was evaluated. (C) Sequences of peptides enriched
during rounds 2–4. The enrichment was calculated based on peptide abundance
from the total number of peptides sequenced. (D) Melanoma cell immunore-
activity against the corresponding antipeptide sera by immunofluorescence.
Anti-GLTFKSL serum recognizes a protein on the cell surface of C8161 mela-
noma cells compared with anti-AEKSSYV, anti-FSGWSTV, anti-VSQRNEL, and
negative control serum. (E) Specific peptide cell surface binding to LECs and
C8161 melanoma cells. Binding of GLTFKSL displayed on phage was evaluated
using the BRASIL method. GLTFKSL phage specifically bound to LECs and C8161
melanoma cells compared with insertless control phage (Fd phage) and human
umbilical vein endothelial cells (HUVECs). Relative transducing units (T.U.) are
displayed as means ±SEMs. (F) Nonpermeabilized and permeabilized LECs and
melanoma cells were immunoreactive against GLTFKSL Abs.
2522
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www.pnas.org/cgi/doi/10.1073/pnas.1424994112 Christianson et al.
recombinant protein array consisting of 37,200 redundant pro-
teins from a human fetal brain cDNA expression library. Each
protein is expressed in duplicate in a grid-like pattern, and a posi-
tive hit is considered unique when a protein is detected in duplicate
on the array incubated with antipeptide serum and not detected in
the array incubated with control serum. The GLTFKSL immune
serum specifically recognized two distinct proteins, which were
sequenced and subjected to protein database searching with
BLAST. Two unique positive protein hits were identified and de-
rived from the same protein–protein PPP2R1A. The sequence of
these two clones had a 100% identity match to the PPP2R1A
protein, with one clone spanning residues 474–589 and the other
one corresponding to residues 457–589 (Fig. S1).
Validation of PPP2R1A Membrane Expression. To verify that the
GLTFKSL Ab recognizes PPP2R1A, the residues 457–589 from
PPP2R1A were expressed and purified. As expected, the purified
PPP2R1A fragment was recognized by the GLTFKSL Ab by
Western blot analysis, whereas a control protein was not im-
munoreactive (Fig. 2A). Additionally, binding of GLTFKSL Abs
to the recombinant PPP2R1A fragment was competitively inhib-
ited by the GLTFKSL peptide (Fig. 2B). GLTFKSL Abs recog-
nized full-length recombinant PPP2R1A protein, whereas control
IgGs were completely nonreactive (Fig. 2C).
Reciprocal immunoprecipitation experiments (Fig. 2D) and
transmission EM (Fig. 2Eand Fig. S2 A–F) with a commercially
available PPP2R1A Ab or the GLTFKSL Ab corroborated our
results and revealed the presence of PPP2R1A on the membrane
surface of C8161 melanoma cells. Interestingly, the PPP2R1A
protein seems to be mainly located on membrane blebs that are
potentially part of the leading edge of migrating melanoma cells
(Fig. S2). To confirm additionally the identity of the PPP2R1A
protein, we silenced PPP2R1A protein expression in C8161 mel-
anoma cells with lentiviral shRNA constructs. Protein knockdown
was confirmed by immunoblotting with either PPP2R1A or
GLTFKSL Abs (Fig. 2F).
Next, to investigate whether PPP2R1A membrane expression
is associated with melanoma metastasis, PPP2R1A cell surface
expression was measured by FACS with a panel of 14 melanoma
cell lines (Fig. 2G). Our analysis revealed a 40% increase in
membrane expression of PPP2R1A in MeWo, UACC 62, C8161,
and WM2664, which correlates with reported PPP2R1A micro-
array differential expression (23). More importantly, PPP2R1A
overexpression correlated with metastatic potential in these cell
lines from their original source (i.e., MeWo LN metastasis). Al-
together, these results indicate that PPP2R1A is the antigen for the
GLTFKSL Ab and further implicate its role in melanoma pro-
gression and metastasis.
Lymphatic–Melanoma Cell Interaction Is Mediated by PPP2R1A. Pep-
tides recovered after the last round of LyV screening aligned with
the primary sequence of PPP2R1A. Several peptides were found
to mimic amino acid stretches in the PPP2R1A protein. In-
terestingly, the GLTFKSL peptide was also found to align near
the C terminus of PPP2R1A (Fig. S3). The fact that GLTFKSL
mimics a linear sequence found in native PPP2R1A protein sug-
gests that we identified a linear epitope. When mapping protein–
protein interactions with combinatorial peptide libraries, linear
epitopes are adjacent residues in the primary protein sequence as
opposed to conformational epitopes that comprise amino acids
that are distant in the primary sequence but brought together in
the native folded conformation. The combination of a putative
linear epitope with (i) GLTFKSL phage binding to LECs and
C8161 melanoma cells and (ii) Abs raised against GLTFKSL
binding the PPP2R1A protein expressed on the cell surface in-
dicated that PPP2R1A may form homodimers that can potentially
mediate a heterotypic cell–cell interaction between the lymphatic
endothelium–metastatic melanoma cell interface.
To investigate whether a homophilic PPP2R1A–PPP2R1A
interaction mediated by the GLT peptide sequence occurs, we
first immobilized GLTFKSL or control peptides on 96-well plates
in a modified ELISA binding assay. In this scenario, GLTFKSL
phage should bind to GLTFKSL peptide. We observed specific
binding of GLTFKSL phage to synthetic GLTFKSL peptide,
whereas no binding was observed to control peptides (CTL1 and
CTL2) or BSA (Fig. 3A). In addition, we immunocaptured native
PPP2R1A from C8161 cell extracts with the commercially available
PPP2R1A or the GLTFKSL Abs. GLTFKSL phage bound spe-
cifically to proteins immunoprecipitated by either the PPP2R1A
Ab or the GLTFKSL Ab compared with a control Ab and
insertless phage (Fig. 3B). Next, binding of the GLTFKSL pep-
tide to C8161 melanoma with reduced PPP2R1A expression
(PPP2R1A shRNA) was reduced by about 50% compared with
nontargeted (NT) control (shRNA) cells (Fig. 3C)withtheBio-
panning and Rapid Analysis of Selective Interactive Ligands
methodology.
To examine further whether PPP2R1A mediates heterotypic
cell–cell interaction, lymphatic cells (HDLECs) were plated and
allowed to form a monolayer. Next, C8161 cells were labeled with
carboxyfluorescein succinimidyl ester (Fig. S4), mixed with the
highest noncytotoxic concentrations of anti-PPP2R1A or CLTFKSL
peptide (previously determined), and incubated at normal growth
conditions. Knockdown (PPP2R1A shRNA) and NT shRNA
control cells were also prepared and evaluated. Melanoma cell
adhesion was significantly impaired when anti-PPP2R1A Ab (10
μg/mL) was added into the coculture (Fig. 3D) compared with
Fig. 2. GLTFKSL peptide is the molecular mimic of PPP2R1A. (A,Left)Ex-
pression of the truncated PPP2R1A 457–589 and a control protein from the
microarray were verified by Coomassie blue staining. (A,Right) Anti-
GLTFKSL Ab recognizes the truncated recombinant PPP2R1A protein by
Western blot. (B) Binding specificity of GLTFKSL Ab to truncated recombi-
nant PPP2R1A. The Ab reactivity against the recombinant protein was inhibi-
ted by addition of GLTFKSL peptide. (C) Anti-GLTF KSL recognizes recombinant
human PPP2R1A by Western blot. (D) Reciprocal immunoprecipitation. Both
anti-PPP2R1A and anti-GLTFKSL immunoprecipitate PPP2R1A from C8161
melanoma cell extracts. (E) Transmission EM of anti-GLTFKSL and anti-
PPP2R1A binding to C8161 cells. Secondary Abs labeled with 18-nm gold
particles were used for detection. (F) Silencing of PPP2R1A by shRNA in
C8161 melanoma cells. Silencing was confirmed by immunoblotting with the
anti-PPP2R1A and anti-GLTFKSL Abs. NT shRNA was used as control vector.
(G) Cell surface binding of anti-PPP2R1A to a panel of human melanoma cell
lines. The percentage of cell surface binding as analyzed by FACS is depicted
as mean ±SD. Rabbit IgG was used as a negative control. IP, immunopre-
cipitation; WB, Western blot.
Christianson et al. PNAS
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MEDICAL SCIENCES
the isotype control. Cell toxicity or difference in cell adhesion of
both HDLECs and C8161 to the substrate (Fig. 3E) was not ob-
served after addition of anti-PPP2R1A. However, similar inhibition
of melanoma cell attachment was observed in PPP2R1A-silenced
C8161 cells (Fig. 3F) or cells treated with the GLTFKSL peptide
(Fig. 3G). PPP2R1A knockdown C8161 cells showed a significant,
approximately twofold reduction in the number of melanoma cells
cultured on a monolayer of HDLECs compared with C8161 cells
transduced with control NT shRNA. No significant changes in cell
proliferation or cell adhesion to the HDLEC monolayer substrate
were observed when C8161 was stably transduced with PPP2R1A
shRNA (Fig. 3F,Right). Addition of GLTFKSL peptide (Fig. 3G)
decreased melanoma cell attachment to lymphatic cells by 20%
relative to control peptide or in the absence of peptide treatment.
Interestingly, melanoma cell migration was significantly inhibited
in the presence of the anti-PPP2R1A Ab (10 μg/mL) or the
GLTFKSL peptide (10 μM). Anti-PPP2R1A Ab significantly
reduced C8161 cell migration by 1.6-fold compared with un-
treated cells (Fig. 3H). This effect was more robust when mel-
anoma cells were incubated with the GLTFKSL peptide (Fig.
3I), reducing cell migration by 2.8-fold. Overall, these data show
that PPP2R1A mediates a heterotypic cell–cell interaction
through homodimer formation.
PPP2R1A Is Accessible in Vivo. Next, localization of the PPP2R1A
protein was assessed in orthotopic C8161 tumor xenografts by
immunofluorescence. Frozen sections of explanted tumors were
coimmunostained with Abs against PPP2R1A, CD34 (a vascular
endothelium protein), and the LyV protein podoplanin. PPP2R1A
was ubiquitously expressed within tumor cells. Moreover, PPP2R1A
extensively colocalized with LECs, whereas colocalization was
not observed in blood vessels (Fig. 4A). To determine whether
PPP2R1A was accessible on melanoma and LECs in vivo, we
evaluated the biodistribution of GLTFKSL phage in C8161
tumors in tumor-bearing mice by immunohistochemistry with
antiphage Abs. We show that the targeted GLTFKSL phage
selectively and specifically bound to melanoma tumors compared
with a control phage and control organs (Fig. 4B). High levels of
PPP2R1A expression were also found on C8161 tumor cells
relative to controls, thus confirming that PPP2R1A is an acces-
sible tumor target in vivo (Fig. 4C).
PPP2R1A Expression on Melanoma Patient Biopsy Samples Correlates
with Poor Outcome. Thirty-one human patient samples with
metastatic melanoma (grade IV) were evaluated to examine the
clinical relevance of PPP2R1A expression on LECs and mela-
noma cells. Melanoma cells as well as LECs were isolated and
subjected to FACS analysis to evaluate cell surface expression.
All 31 patient samples had high (>50%) PPP2R1A cell surface
expression for both cell types. More importantly, a statistically
significant correlation of PPR2RA1 expression between mela-
noma and LECs was found (Pearson correlation; P=0.017) (Fig.
5A). PPP2R1A expression on patient-derived LECs was ranked
from high to low and correlated with the matched melanoma cell
sample from the same patient. A critical issue in protein dis-
covery with combinatorial screenings is to test the putative marker
on subjects that were not included in the original assay design.
Histological analysis of human melanoma samples at different
stages showed high expression of PPP2R1A in primary tumor
samples, in-transit metastasis, and LN metastasis (Fig. 5B).
Our data show that the two-arm approach developed in this
report allows identification of singular and relevant cell surface
receptors. Furthermore, we established that PPP2R1A, a pre-
viously unrecognized cell surface receptor, contributes to cell–
cell interactions between melanoma and lymphatic cells. Finally,
increased expression of PPP2R1A in both lymphatic and tumor
cells during melanoma progression indicates that PPP2R1A may
play an important role during melanoma invasion and metastasis
through the lymphatic vasculature. Additional studies will elu-
cidate and accelerate understanding of cell–cell interactions
within the context of LyV biology and melanoma metastasis.
Discussion
In this report, we show that tailoring combinatorial library se-
lection of phage display to use available patient samples ex vivo
in parallel with traditional in vitro experiments can be success-
fully used to identify potentially clinically relevant tumor bio-
markers. The successful isolation and validation of PPP2R1A as
a candidate biomarker of melanoma metastasis through inter-
actions between melanoma tumor cells and LECs indicate the
potential of this approach. PPP2R1A is the scaffolding subunit A
of the protein phosphatase 2A (PP2A), one of four major serine/
threonine protein phosphatases. PP2A plays an important role in
cell proliferation, death, mobility, cell cycle control, and de-
velopment and is involved in the regulation of numerous signaling
Fig. 3. PPP2R1A mediates the cell–cell interaction between LECs and mel-
anoma. (A) GLTFKSL phage bound specifically to the GLTFKSL peptide
compared with control peptides, BSA, and insertless phage. (B) PPP2R1A was
immunocaptured from C8161 cell extracts with the anti-PPP2R1A and anti-
GLTFKSL Abs. GLTFKSL phage bound to both immunocaptured proteins
compared with control. All relative transducing units (T.U.) were expressed
as means ±SEMs. (C) The Biopanning and Rapid Analysis of Selective In-
teractive Ligands method was used to evaluate binding of GLTFKSL phage to
C8161 cells stably transduced with PPP2R1A shRNA or NT shRNA. Binding of
GLTFKSL phage to PPP2R1A-silenced cells was reduced by about 50%. Insert-
less control phage (Fd phage) served as a negative control. (D)Abagainst
PPP2R1A inhibits cell–cell interaction between melanoma and lymphatic cells.
(E) Lymphatic and melanoma cell adhesion to the ECM was evaluated in vitro
with or without addition of anti-PPP2R1A Ab. (F,Left) Silencing of PPP2R1A in
C8161 decreases melanoma cell attachment to LECs. (F,Right) Cell adhesion to
the ECM was not compromised after PPP2R1A knockdown. (G)Melanoma–
lymphatic cell interaction inhibited by the GLTFKSL peptide. Cell migration is
mediated by surface expression of PPP2R1A. (H) Anti-PPP2R1A Ab. (I)GLTFKSL
peptide. CTL, control. *P<0.05; **P <0.01.
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www.pnas.org/cgi/doi/10.1073/pnas.1424994112 Christianson et al.
pathways (24, 25). PP2A exists in two general forms—a hetero-
dimeric core enzyme and a heterotrimeric holoenzyme. The core
enzyme consists of the scaffolding subunit A and a catalytic sub-
unit C, and each can exist as two isoforms. The PP2A core enzyme
interacts with a large number of alternative forms of the variable
regulatory subunit B to assemble into a holoenzyme, which rec-
ognizes many different substrates (24, 25). Alterations affecting
different subunits or isoforms have detrimental effects on phos-
phatase function and have been shown to promote tumorigenesis
(26, 27). Several mutations of the PPP2R1A isoform have been
reported in breast and lung cancers; ovarian, uterine, and endo-
metrial carcinomas; and malignant melanoma (22, 27–30). Be-
cause loss of PPP2R1A in mice is lethal, at least a minimal amount
of PPP2R1A is required to maintain cell viability (31, 32).
Considering the myriad functions that can be affected by alter-
ations of the PPP2R1A isoform, it is difficult to designate its role
in tumorigenesis, and efforts to reconcile the diverse functions of
PPP2R1A are ongoing (26, 27, 33).
To the best of our knowledge, there are no reports of cell
surface expression of PPP2R1A, its expression in the extracel-
lular environment, or its function other than its activity as
a phosphatase enzyme complex. Our data indicate that extra-
cellular PPP2R1A mediates interactions between LECs and
melanoma cells. Thus, an extracellular function for PPP2R1A in
melanoma through cell–cell interactions with the LECs, as
revealed here, could be relevant for a number of different can-
cers and is possibly independent of the PP2A enzyme. The role
of scaffold proteins in cell adhesion and their recruitment based
on chemical or biophysical signals are well-described, and the
implications of this knowledge in tumor invasiveness, aggres-
siveness, and metastasis formation have been shown (34, 35).
Our findings are consistent with this knowledge and were an-
ticipated with an unbiased combinatorial peptide library in the
phage panning and selection scheme as described above. Proteins
are selected based on their expression profile on the cell surface
and their availability or accessibility to circulating ligands (36).
This approach for identifying and validating ligand–receptor inter-
actions has previously shown a novel extracellular function for the
CRKL protein in tumorigenesis (37). The tumor microenvironment
may induce this heretofore unrecognized, to our knowledge,
Fig. 4. PPP2R1A is expressed on LyVs in vivo. (A) Confocal microscopy was
performed on C8161 tumor frozen sections stained with PPP2R1A (red),
Podoplanin (green), and CD34 (blue). Colocalization (yellow) of PPP2R1A
and Podoplanin is depicted in the merged image. Neg. CTRL, negative
control. (B) Immunohistochemical analysis of GLTFKSL phage homing. Posi-
tive staining (red arrows) is shown in the tumor of GLTFKSL-injected mice,
whereas insertless phage staining in the tumor is negative. Muscle was the
negative control organ. Liver is part of the reticuloendothelial system, and
therefore, phage is present. Representative pictures are shown (magnifi-
cation: 200×). (C) PPP2R1A expression in C8161 tumors. PPP2R1A is highly
expressed in C8161 tumors as shown by immunohistochemistry. CTRL, con-
trol. (Magnification: 200×.)
Fig. 5. PPP2R1A expression on melanoma patient samples. (A) PPP2R1A is
expressed on the cell surface of human metastatic melanoma samples (grade
IV). Flow cytometry analysis of melanoma samples is graphically represented
as a waterfall blot. A positive correlation is observed between expression of
PPP2R1A on the surface of LECs and melanoma cells as calculated by the
Pearson correlation method (r=4, P=0.017). NHEM, normal human me-
lanocyte. (B) Histological analysis of human melanoma samples. Primary
melanoma (Prim. Tumor), in-transit metastasis (In-transit), and LN metastasis
(LN Met) samples stain positive for PPP2R1A. Neg. CTRL, negative control.
(Magnification: 200×.)
Christianson et al. PNAS
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vol. 112
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MEDICAL SCIENCES
functionality, and these results show the biological significance of
these extracellular roles in tumor cell proliferation and migration.
Homodimer formation described here is reminiscent of another
report, where a homophilic interaction of the cell surface glyco-
protein MUC18 was observed between melanoma cells and B-1
lymphocytes (38). It was suggested that the surface expression of
MUC18 may be differentially regulatedonthetwoseparatecell
surfaces and that the interactionofthetwocelltypesthrough
a homophilic MUC18–MUC18 ligand–receptor system influences
melanoma metastasis (38). The fact that both the MUC18 and
PPP2R1A homodimeric protein complexes involve scaffold pro-
teins is unsurprising and indicative of the translational potential for
evaluating scaffold proteins and their cell–cell junctions for markers
of tumor progression and metastasis. Indeed, additional experi-
ments are needed to understand the functional role of PPP2R1A
on the cell surfaces of the LECs and melanoma cells and the
regulatory mechanism that influences its extracellular expression.
Materials and Methods
Patient Samples. This study adheres strictly to current medical ethics rec-
ommendations and guidelines regarding human research, and it has been
reviewed and approved by the Clinical Ethics Service, the Institutional Bio-
hazard Committee, the Clinical Research Committee, and the Institutional
Review Board of The University of Texas MD Anderson Cancer Center.
Animals. Six- to eight-week-old nude mice were purchased from Charles River
or The Jackson Laboratories. All animal work followed standard procedures
approved by The University of Texas MD Anderson Cancer Center.
Statistics. A correlation test was performed with R (The R Project for Statistical
Computing) according to the Pearson correlation method. SEM and SD were
calculated with the GraphPad Prism program.
Additional methods are described in SI Materials and Methods.
ACKNOWLEDGMENTS. This work was supported by awards from the Gillson
Longenbaugh Foundation and the American Recovery and Reinvestment Act
of 2009, a Cancer Center Support grant (to University of New Mexico Cancer
Center), and a Department of Defense grant (to W.A. and R.P.)
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