Oral Oncology 78 (2018) 126–136

Contents lists available at ScienceDirect

Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Review

Podoplanin emerges as a functionally relevant oral cancer biomarker andtherapeutic target

T

Edward P. Retzbacha, Stephanie A. Sheehana, Evan M. Nevela, Amber Batraa, Tran Phia,
Angels T.P. Nguyena, Yukinari Katob, Soly Baredesc, Mahnaz Fatahzadehd, Alan J. Shienbaume,⁎
Gary S. Goldberga,aDepartment of Molecular Biology and Graduate School of Biomedical Sciences, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084, USA
New Industry Creation Hatchery Center, Tohoku University; Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai, Miyagi 980-8575, Japanc
Department of Otolaryngology-Head and Neck Surgery, Rutgers New Jersey Medical School, Newark, NJ 07103, USAd
Department of Diagnostic Sciences, New Jersey School of Dental Medicine, Rutgers University, Newark, NJ 07103 USAe
Department of Pathology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084, USAb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Oral cancer
Oral squamous cell carcinoma
Podoplanin
Signal transduction
Cancer therapy
Biomarkers
Contact normalization
Chemotherapy
CLEC-2

Oral cancer has become one of the most aggressive types of cancer, killing 140,000 people worldwide every year.
Current treatments for oral cancer include surgery and radiation therapies. These procedures can be very effective; however, they can also drastically decrease the quality of life for survivors. New chemotherapeutic
treatments are needed to more eﬀectively combat oral cancer. The transmembrane receptor podoplanin (PDPN)
has emerged as a functionally relevant oral cancer biomarker and chemotherapeutic target. PDPN expression
promotes tumor cell migration leading to oral cancer invasion and metastasis. Here, we describe the role of
PDPN in oral squamous cell carcinoma progression, and how it may be exploited to prevent and treat oral cancer.

The oral cancer burden

Tumors are categorized as T4 if they have invaded another portion of
the mouth or jaw, including but not limited to the cortical bone, ﬂoor of
the mouth, tongue, and the skull base. In addition, numbers of lymph
nodes (N) and distant sites (M) invaded by the tumors are included in
the TNM classiﬁcation [9].
The development of oral cancer is a complex process involving genetic and environmental factors. Major risk factors include tobacco andalcohol use. Indeed, tobacco and alcohol have synergistic eﬀects that
may increase the risk of oral cancer up to 30-fold [10]. The humanpapillomavirus (HPV) has also been implicated in the development oforal cancer, although not as often as more proximal sites such as thepharynx, tonsils, and base of tongue [6].
Over 90% of oral cancers are oral squamous cell carcinoma (OSCC)[5], and these tumors are notoriously resistant to chemotherapeutic
agents. Decades of research with a variety of compounds including alkylating agents, tubulin disruptors, and anthracyclines have failed tosigniﬁcantly increase patient survival or quality of life [11]. Therefore,
surgery and radiation therapy are primarily used to treat oral cancer
patients. These procedures can extend survival rates; however, they alsoOver 14 million new cancer cases are diagnosed each year, whichkill a person about every 5 seconds around the world [4]. These statistics indicate that many cancers are not treated successfully. Oralcancer has earned its place among the world’s most vicious malignancies. Oral cancer kills over 8,000 people in the USA and 140,000
people worldwide every year, and these numbers are rising [5,6].
Sites for oral cavity cancer include the lips, tongue, ﬂoor of themouth, upper and lower alveolar ridge, retromolar trigone, buccal
mucosa, and hard palate. Precancerous and early presentations areoften found as white or red mucosal lesions deﬁned as oral leukoplakia(OLP) or erythroplakia, respectively. Precancerous lesions proceed fromhyperplasia to dysplasia and carcinoma in situ before developing into
invasive malignancies [7,8].
Oral cancer is categorized into four groups according to their tumor,
node, and metastasis (TNM) stages. Tumor stages T0, T1, T2, and T3
cases are classiﬁed as having no evidence of tumor, tumors less than orequal to 2 cm, greater than 2 cm, or greater than 4 cm, respectively.

⁎

Corresponding author.
E-mail address: gary.goldberg@rowan.edu (G.S. Goldberg).

https://doi.org/10.1016/j.oraloncology.2018.01.011
Received 17 November 2017; Received in revised form 14 December 2017; Accepted 18 January 20181368-8375/ © 2018 Elsevier Ltd. All rights reserved.

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process, and can be used to identify premalignant lesions that arebound to develop into oral cancer. Analysis of over 300 premalignant
oral lesions pooled from several retrospective studies [7,33–36] issummarized in Fig. 2a. These data indicate that over 46% of lesions
with notable PDPN expression progressed to OSCC. In contrast, lessthan 14% of lesions without notable PDPN expression progressed to
OSCC. Thus, PDPN expression increases the probability of OSCC formation from histologically benign lesions by over 3-fold. However, thisnumber could be an underestimate of PDPN involvement since more
suspicious lesions are most likely to be slated for biopsy by health care
professionals. For example, data from some studies indicate that over
80% of oral leukoplakias that express high levels of PDPN convert tooral cancer [7].
In addition to malignant progression, PDPN expression also correlates with OSCC mortality. As shown in Fig. 2b, 5 year overall anddisease free survival drops from 86% and 100% for patients with tumors with undetectable PDPN expression levels to 23% and 37% forpatients with tumors exhibiting high PDPN expression [2,3]. These data
indicate that PDPN expression leads to a 3 to 4-fold decrease in 5 year
survival for oral cancer patients.
PDPN is expressed in many forms of cancer. As with OSCC, PDPN
expression has been shown to promote mammary carcinoma [37,38],
glioma [39], other types of squamous cell carcinoma (SCC) [7,8],
melanoma [40–42], ovarian cancer [43], and pulmonary adenocarcinoma [44,45]. Indeed, PDPN appears to play a key role in fundamental
mechanisms leading to tumor invasion and metastasis.
PDPN plays a unique role in the tumor microenvironment. In addition to tumor cells, PDPN is expressed in inﬁltrating cancer associatedﬁbroblasts (CAFs). Results from several reports indicate that CAFs utilize PDPN to increase motility and survival of neighboring tumor cells,
particularly lung adenocarcinoma and melanoma cells [41,44,45]. Xenograft human OSCC cells induce PDPN expression in mouse host inﬁltrating CAFs in experimental models as shown in Fig. 3. Lymph node
stromal cells that express PDPN have also been shown to inhibit the
proliferation of T helper cells in order to promote melanoma progression [46,47].

Table 1
Examples of tumor suppressors and promoters altered in oral cancer.
Suppressor

Function

References

Bax
E-Cadherin
p16
p21
p27
p53

[172]
[16]
[23]
[20]
[172,173],[20,22]

P120/catenin
Rb

Apoptotic activator
Homotypic adherins junction
CDK4/6 inhibition
CDK1/2 inhibition
Cyclin E – CDK2 inhibition
Cell cycle arrest, genome protection, andapoptosis activation
Cell adhesion signaling
Cell cycle arrest

Promoter

Function

References

Bcl-2
CD133
Cyclin D1
EGFR
Ki-67
MMP-2/9
PDPN
Src
Wnt

Apoptotic modulator
Hematopoietic stem cell marker
CDK activator
Receptor tyrosine kinase
Cell proliferation marker
Extracellular matrix degradation
Cell motility
Nonreceptor tyrosine kinase
Migration, diﬀerentiation[172,176,177][25][21,23,178–180][19,22,26,181]
[182]
[183]
[27]
[25,27][24][17,18][23,174,175]

cause disﬁgurations and sequelae that drastically decrease the qualityof life for survivors. These patients often experience diﬃculty in airway
management, speech, and mastication [12,13]. A better understandingof mechanisms leading to OSCC is needed to develop more eﬀective
methods to detect and treat oral cancer.
A number of tumor promoters and suppressors have been identiﬁed
in OSCC progression, some of which are shown in Table 1. Activities oftumor suppressors including p53 [14,15], E-cadherin [16], and p120catenin [17,18] are often disrupted in OSCC cells, while the activities oftumor promoters such cyclin D1 [19–23], Wnt signaling pathways [24],the EGFR and Src tyrosine kinases [25,26], and transmembrane receptor podoplanin (PDPN) [27] are often increased. In particular, PDPN
has emerged as a functionally relevant biomarker and potential chemotherapeutic target to prevent and treat oral cancer.

PDPN in development and disease
The emergence of PDPN
As with many tumor promotors, PDPN plays critical roles in ontogeny [48]. PDPN is expressed early in development, starting with the
proepicardial organ in the embryonic foregut [49]. Studies with PDPN
knockout mice demonstrate that it goes on to play key roles in thedevelopment of the heart, lungs, and lymphatic system [50].
PDPN associates with the C-type lectin-like 2 (CLEC2) receptor onplatelets as described below (see Fig. 4) [51]. This association plays acritical role in the genesis of the lymphatic system. Presumptive lymphatic vessels sprout from the cardinal vein during mammalian development. Platelets that express CLEC2 interact with PDPN on these

Aggregate analysis of over 1,000 samples indicate that PDPN israrely expressed in normal oral epithelial cells, but is found in over 50%
of oral cancers [2,3,27–31]. Moreover, PDPN expression in oral cancers
is likely to be much higher than actually reported. This is because PDPN
expression is found mainly in the invasive fronts of oral cancers. For
example, PDPN is clearly seen at the invasive front of OSCC as shown in
Fig. 1a. Indeed, we [1] and others [32] found PDPN expression in
100% of OSCC examined in focused studies.
PDPN expression is induced very early in the OSCC transformation

Fig. 1. PDPN expression in human oral cancer. (a)Stage 1 OSCC from the tongue of a 61 year old
male. (b) Oral leukoplakia from the tongue of a
47 year old male. Bar = 40 µm.

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Fig. 2. PDPN expression and oral cancer mortality. (a) PDPN expression correlates with transformation of premalignant oral lesions to OSCC. Data are shown as mean + SEM (n = 194
and 135 for PDPN negative and positive lesions, respectively) [7,33–36]. (b) PDPN expression inversely correlates with overall and disease free 5 year patient survival. Data are shown asmean + SEM (n = 143 and 63 for overall and disease free survival, respectively) [2,3].

and long term synaptic potentiation. Data from these experiments alsosuggest that PDPN interacts with NGF (nerve growth factor) and may
play a role in the development of Alzheimer's and other neurodegenerative diseases [62]. Interestingly, PDPN and amyloid precursor protein (APP) are both cleaved by presenilin-1/gamma-secretase [63].
PDPN is expressed in kidney podocytes that maintain selective
glomerular ﬁltration [64]. Decreased PDPN expression in kidney podocytes results in ﬂattening of foot processes, causes proteinuria, andhas been reported in nephrosis [64,65]. PDPN also appears to enablekidney podocytes to activate AKT kinase and avoid apoptosis in response to angiotensin signaling [66,67].
PDPN appears to play a key role in adaptive immunity and inﬂammatory disease. Dendritic cells express CLEC2, which associateswith PDPN ﬁbroblastic reticular cells to increase lymph node expansion
and promote innate immunity [50,68]. Increased PDPN expression isfound in chondrocytes and synovial ﬁbroblasts associated with rheumatoid and osteoarthritis [69–75] In addition, PDPN is expressed on
inﬂammatory dermal ﬁbroblasts that induce interleukin 17 production
by neighboring T helper cells to promote psoriasis [76]. PDPN is also
found in general lymphatic expansion related to inﬂammation. For
example, PDPN is found in lymphatic vessel formation that occurs in
the intestines of patients with ulcerative colitis and Crohn's disease[77].
Taken together, data indicate that PDPN plays key roles in development and homeostasis. In particular, PDPN promotes signaling events
that promote cell motility and inﬂammation which are both fundamental hallmarks of cancer [48]. Indeed, the vast majority of oral

future LECs (lymphatic endothelial cells). This interaction causes lymphatic sacs to pinch oﬀ and separate from cardinal veins. This separation is crucial for the lymphatic system to arise from the embryoniccardiovascular system [49], which continues beyond development suchthat PDPN is used as an accepted marker for lymphatic endothelial cells(LECs) [52].
PDPN expression in type 1 alveolar cells is critical for proper lungdevelopment. This process is evidenced by abnormal diﬀerentiation andmorphology of the distal lung in PDPN knockout mice. These micedisplay defective lymphatic systems, but usually die shortly after birthfrom respiratory failure [53–55]. PDPN null mice also suﬀer from heart
deformation resulting from abnormal proepicardial organ morphology,
inhibited migration of epicadium-derived cells, and abnormal pulmonary vein development [56,57]. PDPN is also expressed through thedeveloping heart lymphatics, which allow for drainage of the subepicardium and the myocardial wall [58]. PDPN expression in heartdevelopment, as well as the vascular system, also plays a critical role incontrolling venous thromobembolism and deep vein thrombosis, which
may be another area where PDPN can become a clinically relevanttarget [59,60].
While PDPN knockout mice are considered to be embryonic lethal, asmall percentage of these animals survive to adulthood without obvious
phenotypic abnormalities. This phenomena may result from redundant
receptor signaling systems. However, these PDPN null survivors provide
a unique model to study PDPN functions in adult mice [61]. For example, recent studies indicate that PDPN expression in the dentate
gyrus of the hippocampus aids in memory formation, spatial learning,

Fig. 3. OSCC cells induce PDPN expression in inﬁltrating host murine cancer associated ﬁbroblasts inxenograft tumors. Tumors from human OSCC cells
were examined with antisera speciﬁc for human (D240, green) and mouse (8.1.1, red) PDPN by immunohistochemistry as previously described [1].

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Fig. 4. PDPN topology and binding partners.
Podoplanin contains an amino terminal signal
peptide (white), 4 extracellular PLAG domains(pink), transmembrane region (lavender), andintracellular carboxyl tail (solid gray). Conﬁrmed
and potential glycosylation sites are indicated by
red and green hexagons, respectively. A GxxxG
motif in the transmembrane region may drive
PDPN to form a homodimer and traﬃc to lipid
rafts. CLEC2, HSPA9, CD44, Galectin 8, andCCL21 bind to extracellular PDPN domains.
CLEC2 interaction sites have been localized to
PDPN residues G45, D48, and D49, with the aid ofglycosylation at T52, along with E81, D82, andT85 in a recently deﬁned PLAG4 domain (E81E87). The integral membrane protein tetraspanin
CD9 interacts with the PDPN transmembrane domain. The ERM proteins ezrin and moesin interact
with the PDPN intracellular tail to modulate
pathways involving ROCK/LIMK activities, aswell as actin polymerization by Rho GTPases. The
PKA and CDK5 kinases can phosphorylate intracellular serines to inhibit cell motility. Figure isbased on human PDPN with relevant amino acids
indicated by number but not drawn to scale.

cancer treatment complications and deaths results from tumor invasion
and metastasis [5,6]. PDPN structural and functional investigations canlead to a greater understanding of how these events can be treated as
discussed below.

Table 2
Podoplanin nomenclature.

PDPN nomenclature, regulation, and structure
Podoplanin has been independently discovered and named by manygroups over the past thirty years. A chronological list of these names isshown in Table 2. PDPN was ﬁrst detected in human adenocarcinoma
cells, fetal testis, seminomas, and dysgerminomas with the rabbit
monoclonal antibody called “M2A” in 1986 [78]. PDPN was then discovered as a marker for type 1 alveolar cells and named “T1α” in 1988
[79]. PDPN was also found on mouse colon adenocarcinoma cells as a
44 kD glycoprotein called “gp44” in 1988 [80]. PDPN was then discovered again as a gene expressed by 12-O-tetradecanoylphorbol-13acetate (TPA) and Ras transformed mouse osteoblastic cells and named“OTS-8” in 1990 [81]. The term “podoplanin” was ﬁrst used upon
discovering its role in promoting ﬂattened foot processes in rat

Name

SourceYear

References

M2A
T1α
GP44
OTS-8

Human ovarian adenocarcinoma cells
Rat type 1 epithelial cells
Mouse colon adenocarcinoma
TPA and Ras transformed mouse osteoblastic
cells
Mouse lymphoid tissue
Rat osteosarcoma
DMBA transformed mouse keratinocytes
Rat glomerular cells
Human lung cDNA expressed in Xenopus
oocytes
Human placenta
Human oncofetal and germ cell tumors
Mouse embryonic carcinoma and central
nervous system
Human colorectal tumors
Rat lungs exposed to inﬂammatory agents

1986
1988
1988
1990

[78]
[79]
[80]
[81]

1992
1996
1997
1997
1998

[184]
[185]
[186]
[64]
[187]

1998
1999
2002

[188]
[189]
[190]

2003
2006

[191]
[192]

GP38
E11
PA2.26
Podoplanin
T1α-2
GP36
M2A/D2-40
RANDAM-2
AggrusrT140

129

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glomerular cells in 1997 [64]. “Podoplanin” and “PDPN” have become
the oﬃcially accepted name and symbol as used throughout this review.
PDPN presents some complex regulatory considerations. The PDPN
gene is located on chromosome 1 in humans (loci 1p36.21) and chromosome 4 in mice (loci 4E1). The gene is rather large at about 600,000
bases with 8 exons in humans and 200,000 bases with 6 exons in mice,compared to its mRNA transcript sizes of between 2,646 and 2,828
bases (4 variants) and about 1,800 bases (2 variants) in humans andmice, respectively [82,83]. PDPN is translated into a 162 amino acid(18 kD) primary translation product in humans and a 172 amino acid
(19 kD) product in mice [84,85].
Several miRNAs (microRNAs), including miR-29b, miR-125a, andmiR-363, target the 3′ PDPN mRNA region to decrease PDPN expression. miR-29b decreases PDPN expression in kidney podocytes, which
contributes to angiotensin II induced apoptosis [66]. Both miR-29b andmiR-125a suppress PDPN expression to inhibit glioblastoma invasion[39], while miR-363 has been shown to inhibit head and neck squamous cell carcinoma metastasis [86].
Epigenetic regulation of PDPN expression is complex. For example,
promoter methylation and histone hypoacetylation increased PDPN
expression in osteosarcoma cells [87], while promoter methylation inhibited PDPN expression in glioblastoma cells [88]. In addition, histone
deacetylase activity increased PDPN expression in OSCC cell lines (HSC2, HSC-3, Scc-10, and Ca9-22), while there was no signiﬁcant evidenceof PDPN promoter methylation in these cells [31].
Several transcription factors have been shown to regulate PDPN
expression. The PDPN promoter region contains Prox1 binding sites,
which increases PDPN expression. This is particularly important for
lymphangiogenesis during development. Binding sites for other transcription factors including AP-2, AP-4, C/EBPβ, NF-1, and NFĸB sites
are also been found in the PDPN promoter, and warrant further study[87,89]. For example, NFĸB expression correlates with lymphangionesis and podoplanin expression in cancers including esophageal SCC[90]. TPA and PI3K/Akt activity leads to AP-1 upregulation of PDPN
expression in mouse skin cancer and human glioblastoma, respectively[88,91]. Sox2 can also increase PDPN expression in cancer stem cells[92]. Other factors have also been implicated in PDPN expression
during cancer progression. These include Sp1 and Sp3 in osteosarcoma
cells [87], as well as combined FGF2/BMP4 signaling in neural stem
cells [93]. Most pertinent to this review, epidermal growth factor(EGFR) activation of STAT3 [94], TGFβ [31,95], and Epb1 activity [96]can increase PDPN expression in cancers including OSCC.
PDPN contains an amino terminal signal peptide followed by extracellular platelet aggregation (PLAG) domains, a transmembrane region, and a short intercellular carboxyl tail as shown in Fig. 4. The
transmembrane region contains glycine based motifs that may drive
PDPN to form dimers in the cell membrane [97]. The transmembrane
and intracellular PDPN domains consist of about 30 amino acids [64],which leave over 80% of the protein outside of the cell where it caninteract with extracellular modulators.
PDPN contains 25 potential glycosylation sites along the extracellular region of the protein [98,99]. The primary PDPN translation
product is modiﬁed from an 18–20 kD polypeptide into a mature protein with an apparent molecular mass in the 40 kD range. These modiﬁcations include galactose and N-acetylgalactosamine with α2-3 andα2-6 linked sialic acid glycosylation events which contribute to protein
interactions and signaling events [84,100,101].

cyclin dependent kinase 5 (CDK5) as shown in Fig. 4.
CLEC2 interaction sites have been localized to PDPN residues G45,
D48, and D49, with the aid of glycosylation at T52 [102,103], alongwith E81, D82, and T85 in a recently deﬁned PLAG domain (E81-E87)[104]. This association activates Src dependent CLEC2 phosphorylationand directs platelet aggregation during lymphatic development andmetastasis by tumor cells that express PDPN [105]. Indeed, antiserathat block CLEC2 interactions with PDPN can signiﬁcantly inhibit
tumor growth and metastasis. In addition, dendritic cell migration canalso be linked to PDPN-CLEC2 interactions [106].
The integral membrane protein tetraspanin CD9 interacts with the
PDPN transmembrane domain. CD9 is a tetraspanin glycoprotein receptor that mediates a variety of events associated with cell adhesion,
migration, and platelet aggregation. However, glycosylated tetraspanin
CD9 appears to oppose the action of CLEC2, binding podoplanin to
inhibit platelet aggregation and lung tumor metastasis [107].
The hyrularonan receptor CD44 is a widely expressed integral
membrane protein that interacts with podoplanin. Like PDPN, CD44 isalso a functionally relevant cancer biomarker that is found in many
types of cancer including oral squamous cell carcinoma [108]. CD44
associates with PDPN to induce epithelial mesenchymal transition, lamellipodia formation, cell adhesion, and directional cell migration[109]. PDPN and CD44 also colocalize in microvesicles and exosomesthat traﬃc between cells to induce epithelial mesenchymal transition(EMT) and lymphatic capillary morphogenesis [110].
PDPN interacts with soluble factors including Gal8 (galectin 8),
CCL21 (C-C motif chemokine 21), and HSPA9 (heat shock protein A9)
in addition to integral membrane proteins such as CLEC2 and CD9. This
interaction is exempliﬁed by Gal8. Gal8 is a mammalian lectin that isfound on lymphatic and vascular endothelial cells. Gal8 interacts withglycosylated receptors on extracellular matrix proteins, integrins, andPDPN. In particular, Gal8 targets PDPN to promote lymphatic endothelial cell adhesion, migration, and lymphatic vessel formation[111]. The interaction between galectin 8 and PDPN also induces inﬂammatory lymphangiogenesis precipitated by immune responses[112].
CCL21 is secreted mainly by lymphatic endothelial cells, and by anumber of other cell types to a lesser extent. CCL21 targets C-C chemokine receptor type 7 (CCR7) chemokine receptors to promote directional T-cell migration. However, CCL21 is also produced by, andtargets PDPN on, ﬁbroblast reticular cells to promote diﬀerentiation ofregulatory T (Treg) cells in the thymus [113]. Interestingly, OSCC cells
also produce CCL21 to promote lymph node invasion with the help ofrecruited macrophages [114]
HSPA9 is a cognate member of the heat shock family of proteins thatis found in the extracellular matrix, mitochondria, and cytoplasm ofvariety of cells. HSPA9 associates with proteins including p53 and
EGFR in addition to PDPN. HSP mutations lead to development ofsideroblastic anemia and even-plus syndrome (characterized by epiphyseal and vertebral dysplasia). In addition, aberrant HSPA9 expression is associated with cancers including glioma and OSCC where it iscolocalizes with PDPN at the tumor stromal interface [115].
The intracellular tail of PDPN contains two serine residues that canbe phosphorylated by PKA and CDK5. These phosphorylation events
eﬀectively decrease PDPN mediated cell motility [41,116]. The PDPN
intracellular tail also contains basic arginine and lysine residues thatinteract with the ezrin/radixin/moesin (ERM) protein family members
ezrin and moesin [85]. This interaction activates small GTPases RhoA
and Rac1, and proteins kinases including Rho associated protein kinase(ROCK) and LIM kinase (LIMK). These events induce actin polymerization and rearrangement to promote cell migration, invasion, andlymphatic capillary morphogenesis [117,118]. These relationships
present clinical relevance since antibodies and lectins can target PDPN
to inhibit Cdc42 GTPase activity, motility, and viability of OSCC cells[1]. In fact, PDPN is among the ﬁrst proteins to be expressed in thetransition from benign to malignant tumor progression as discussed

PDPN binding partners
PDPN is a unique protein without intrinsic functional domains or
enzymatic activities. Thus, PDPN appears to interact with other proteins to aﬀect cell behavior. These binding partners include CLEC2, heatshock protein A9 (HSPA9), CD9, CD44, Galectin 8, Chemokine (C-C
motif) ligand 21 (CCL21), ezrin, moesin, protein kinase A (PKA), and130

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papillary thyroid carcinoma and nodular lymphocyte-predominant
Hodgkin lymphoma [116,124,149]. VEGFR2 (also known as KDR) is
expressed mainly by vascular endothelial cells, as well as many types ofcancers including glioma, breast, and lung carcinoma. VEGFR2 is atyrosine kinase receptor that promotes tumor cell proliferation, migration, and angiogenesis. VEGFR2 expression has become a prognostic
tool to predict OSCC aggression and metastasis [150,151]. Chemotherapeutic drugs targeting VEGFR2 and its VEGF ligands have beendeveloped to inhibit tumor progression and angiogenesis. These drugs
include bevacizumab (Avastin), sunitinib (Sutent), and sorafenib(Nexavar). Eﬀects of these drugs on oral cancer are currently being
evaluated. Clinical trials are being conducted to study the eﬃcacy ofcytotoxic drugs in combination with Bevacizumab for head and neck
SCC (HNSCC) patients [152,153]. Combination therapy utilizing sorafenib and radiation is also being evaluated as a treatment for OSCC
patients [154–156].
In addition to VEGFR2, PDPN was identiﬁed as a tumor promoter
that is upregulated during transformation and reduced by contact
normalization. As discussed throughout this review, PDPN is highly
expressed in many types of cancers including OSCC where it promotes
tumor progression and metastasis. Studies indicate that antibodies andlectins can be used to target podoplanin in OSCC as described below.

below.
Contact normalization
Nontransformed cells form junctions with adjacent cells that force
them to assume a normal phenotype and morphology. This process iscalled contact normalization [116,119]. Clinical implications of contact
normalization can be traced back as early as 1839 [120]. These studies
report cancer recurrence after 15 years or more of disease-free survival,suggesting transformed cells can adopt a normal phenotype and residedormant in the body for many years [119–121]. Contact normalization
plays a chief role in the suppression of tumor cells during early stages oftumorigenesis. Tumor cells must escape contact normalization before
they become malignant and metastasize [116].
Cadherin and connexin interactions promote contact normalization[116,122–124]. Cadherins are calcium dependent transmembrane glycoproteins that form adherens junctions to mediate intercellular adhesion [116,125]. “Classical” cadherins include E-cadherin (epithelial
cadherin) and N-cadherin (neural cadherin). Both E-cadherin and Ncadherin have been identiﬁed as biomarkers in the progression of oral
squamous cell carcinoma. Decreased E-cadherin expression is associated with increased OSCC malignancy and metastasis [126–129].
Conversely, inhibition of N-cadherin expression is associated with decreased OSCC malignancy and metastasis [127,130]. Replacement of Ecadherin with N-cadherin expression in tumor cells is known as the“cadherin switch” during the process of EMT [131]. Carcinoma cells
often become more motile, invasive, and metastatic following thisprocess [132,133].
As with cadherin interactions, gap junctions are also often disrupted
in tumor cells. Connexin proteins form gap junctions to mediate directcommunication between adjacent cells. Gap junctions enable neighboring cells to share intracellular signals including metabolites and ionsin a controlled fashion. Connexin proteins interact with a number ofpartners including cadherins. Similar to cadherins, connexins can act astumor suppressors. Repressed connexin expression has been found incancers including lung, glioma, breast [134–137], and OSCC [138,139].
However, while gap junctions appear to enhance the ability of normal
cells to control the growth of tumor cells, they are not required forcontact normalization [122].
Approximately 0.1% of the transcriptome is aﬀected by contact
normalization. About 60% of these genes are considered tumor suppressors since they are suppressed in the transformed cells but increased by contact normalization, and 40% are considered tumor promoters with increased expression in transformed cells and decreased
expression during contact normalization. These tumor suppressors include four and a half LIM domains (FHL1) and serum deprivation response protein (SDPR), while tumor promoters include transmembraneprotein 163 (TMEM163), vascular endothelial growth factor receptor 2(VEGFR2), and PDPN [116,140].
FHL1 belongs to a family of LIM only proteins that function tocontrol multiple cellular processes including proliferation, diﬀerentiation, apoptosis, adhesion, migration, transcription, and signal transduction [116,141,142]. Decreased FHL1 protein and mRNA expression
is observed in OSCC, which result from DNA methylation of its promoter region [143]. SDPR, a calcium-independent phospholipid
binding protein, acts to induce caveolae formation jointly with caveolin, when phosphorylated by protein kinase C (PKC) and binding
with polymerase I and transcript release factor (PTRF) [144,145]. SDPR
expression may be regulated by FHL1, and decreased expression isobserved in OSCC and many other cancers including breast, kidney, andprostate [146,147].
Tumor promoters involved in contact normalization are of particular interest since they may serve as drug targets. The receptor proteins TMEM163, VEGFR2, and PDPN rise to the top of this list [148].
TMEM163 is a transmembrane protein involved in glutamate transport.
Increased Tmem163 expression has been found in cancers including

Treatments targeting PDPNPDPN has emerged as a clear chemotherapeutic target for oral
cancer. Consequently, compounds that target PDPN are being developed as anticancer reagents. These include antibodies, cell based immunotherapies, and chemical biologics.
Antibodies against PDPN have shown promise as anticancer agentsin preclinical studies. This work began with the NZ-1 monoclonal antibody which targets the PLAG domains of human podoplanin. Initially,
NZ-1 was found to inhibit platelet aggregation to glioma cells mediatedby PDPN-CLEC2 interactions [157]. Further experiments revealed that
NZ-1 and its rat-human chimeric derivatives [158,159] can also inhibit
the growth of glioma [160], mesothelioma [161], and, of interest forthis review, OSCC cells [1]. For example, NZ-1 caused a 30-fold reduction in tumor weight compared with control tumor growth in a
xenograft orthotopic mesothelioma model in SCID mice dosed at 100 µg
i.p. twice a week for two weeks [159]. A mouse-human chimeric antibody against human PDPN, chLpMab-7, was also shown to inhibit
metastasis in a pulmonary xenograft mouse model [162]. Other antiPDPN monoclonal antibodies such as murine and humanized MS-1 also
blocks PDPN-CLEC2 interactions and can eﬀectively inhibit lung tumor
growth by IV administration [103]. Genetically modiﬁed chimeric antigen receptor (CAR)-transduced T-cells that target PDPN have also
been shown to inhibit glioblastoma progression in mouse models. For
example, human T cells expressing modiﬁed NZ-1 injected into the tailvein 7 days after glioblastoma implantation caused a 3-fold reduction intumor growth and 140% longer survival time than controls [163].
Intriguingly, the glycosylation pattern of PDPN appears to change
when expressed by cancerous cells, leading to the development ofcancer speciﬁc monoclonal antibodies (CasMabs) [164]. These highly
speciﬁc reagents oﬀer enhanced potential to target PDPN on cancer
cells. For example, the CasMab chLpMab-2 reacts with glioblastoma,mesothelioma, and lung cancer cells [165]. This technology was also
used to develop chLpMab-23, which can inhibit glioblastoma and OSCC
growth [166]. For example, human OSCC cells that were subcutaneously injected into nude mice, followed by 100 µg of i.p. injected
ChLpMab-23 six times over a thirty-ﬁve-day period showed a 3.5-folddecrease in tumor weight compared to controls. This precursor of thisCasMab antibody, LpMab-23, also shows minimal binding to surrounding lymphatic endothelial cells which express podoplanin [167]as shown in Fig. 5. These cancer speciﬁc PDPN antibodies oﬀer minimal
oﬀsite targeting to present a double-edged tool that can be used to
exploit PDPN as a biomarker with reduced false positives due to131

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D2-40

LpMab-23

OSCC

Fig. 5. Cancer speciﬁc monoclonal antibodies(CasMabs) can speciﬁcally detect PDPN on OSCC cells.
Human clinical samples from a patient with OSCC andoral leukoplakia OLP were examined with antibodies
directed against total PDPN (D2-40) and cancer speciﬁc PDPN (LpMab-23) as indicated. PDPN staining isevident in OSCC cells and OLP with presumptive OSCC
potential by both antibodies, while only D2-40 stained
PDPN on lymphatic endothelial cells (indicated by arrows). Bar = 80 µm.

OLP

Acknowledgments

expression in normal tissues, and also as potential chemotherapeutic
agents with minimal side eﬀects. For example, as shown in Fig. 5, OSCC
cells and oral leukoplakia cells that seem bound to evolve into OSCC areboth recognized by LpMab-23. This reagent may provide a tool that canbe used for early detection needed to formulate best care plans for bestcare treatment.
Podoplanin can also be targeted with other biological chemicals,
exempliﬁed by Maackia amurensis seed lectin (MASL) [1,40]. This
compound is actually a coincidental component of traditional medicines used to treat cancer that have been prepared from the Maackia
amurensis for centuries [168,169]. Comparison between MASL and NZ1 indicate that both inhibit OSCC cell migration and viability at similarnM and µM concentrations, respectively. However, MASL, which binds
terminal sialic acid residues on PDPN [170], can be orally administered
to treat cancer and dynamically targets OSCC cells within minutes of
exposure [1,171]. This potential for topical and systemic treatments
could be particularly useful for oral cancer and precancerous lesions
that are destined to evolve into OSCC.

This work was supported in part by funding from the Osteopathic
Heritage Foundation, Northarvest Bean Growers Association, New
Jersey Health Foundation, and Sentrimed to GSG, the Platform for Drug
Discovery, Informatics, and Structural Life Science from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of Japan,
by Regional Innovation Strategy Support Program from MEXT of Japan,
by Grant-in-Aid for Scientiﬁc Research from MEXT of Japan to YK, andthe New Jersey Health Foundation to AJS.
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Conclusion
Oral cancer is a devastating malady that aﬀects millions of people
and their families. The lack of eﬀective chemotherapeutics is a paramount problem facing this patient population. PDPN has emerged as afunctionally relevant biomarker and chemotherapeutic target. Reagentsare being developed to speciﬁcally target this extracellular receptor on
cancer cells while minimizing oﬀ target chemotherapeutic side eﬀects.
The oral health community is now poised to take advantage of thesetechnologies to better detect, treat, and prevent oral cancer.

Conﬂict of interest
Dr. Goldberg received funding from Sentrimed Inc which holds intellectual property for reagents that target PDPN to combat cancer.
Other authors declare no conﬂict of interest.

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