Tumor Biol. DOI 10.1007/s13277-014-2266-5
REVIEW
Podoplanin—a novel marker in oral carcinogenesis Niharika Swain & Shwetha V. Kumar & Samapika Routray & Jigna Pathak & Shilpa Patel
Received: 19 April 2014 / Accepted: 19 June 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Podoplanin, a transmembrane sialoglycoprotein, is a specific marker for lymphatic endothelial cells which in recent years has gained prominent notoriety for its role in tumor progression and metastasis. It is an extensively studied biomarker for predictive assessment of malignant transformation as well as biologic behavior in both human precancer and cancer, respectively. This review summarizes the association of podoplanin overexpression in oral potentially malignant disorders and oral cancer with special emphasis on its putative role in carcinogenesis as well as its prospective use in targeted therapy. Keywords Podoplanin . Biomarker . Carcinogenesis . Oral potentially malignant disorder . Oral cancer . Epithelial mesenchymal transition
Introduction Podoplanin, though initially detected in murine osteosarcoma cell lines and lymphatic endothelial cells (LECs), was so coined due to its peculiar expression in podocytes or foot processes of renal corpuscles and its potential influence in maintaining the unique shape of podocytes [1]. Subsequently, several PDPN like homologous proteins such as, the oncofetal antigen M2A recognized by the D2-40 antibody and the type I alveolar cell marker hT1α-2 and gp36, a sialoglycoptrotein of N. Swain (*) : S. V. Kumar : J. Pathak : S. Patel Department of Oral Pathology, MGM Dental College and Hospital, 304, Siddhi Vinayak Residency Sector 9, Plot no. 40 Kamothe, Navi Mumbai 410209, Maharashtra, India e-mail: [email protected] S. Routray Department of Oral Pathology, Institute of Dental Sciences, Siksha ‘O’ Anusandhan University, Bhubaneswar, Odisha, India
vascular endothelium and alveolar epithelium, were indentified in human body [2]. PDPN primarily belongs to the family of type 1 transmembrane sialomucin-like glycoproteins. It consists of 162 amino acids, with an extracellular domain rich in serine and threonine residues, a single hydrophobic membrane spanning domain, and a short cytoplasmic endodomain. The extracellular domain is composed of repetitive mucin sequences with extensive O-glycosylated Ser and Thr residues ensuing in an extended rigid structure and a net negative charge. The extremely short cytoplasmic domain composed of only nine amino acids having a functional motif, i.e., Ser (Ser 167), which is a potential cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylation site and a cluster of highly conserved basic amino acids, recruiting site for proteins of the ezrin, radixin, and moesin (ERM) family [3, 4]. Apart from being a prominent lymphatic endothelial cell marker, PDPN has been reported to have a wide cellular distribution such as osteocytes, osteoblasts [5], odontoblasts [6], enamel epithelia, mesothelial cells [7], epidermal basal layer cells [8], salivary myoepithelial cells [9], choroid plexus epithelial cells [10], thymus type 1 epithelial cells [11], prostate myofibroblasts [12], follicular dendritic cells [13], immature cells like fetal germ cells, and developing Sertoli cells [2, 14]. Physiologically, PDPN seems to have multifaceted role in embryogenic development and growth of various organs like lymphatic system, lungs, and heart in humans. In vasculogenesis, PDPN has a crucial role in the separation of lymphatic from the blood circulatory system. The interaction of platelet and PDPN on lymphatic endothelial cells induces platelet aggregation and prevents blood from flowing into new lymphatic vessels budding from the cardinal vein. Furthermore, continued expression of PDPN into adulthood reinforces its importance in maintaining proper lymphatic architecture. In embryogenesis of the heart and lungs, the tissuespecific intrinsic role of PDPN in cell differentiation and
Tumor Biol.
proliferation is speculated in various studies [15–17]. In addition to its physiological role, PDPN has also been implicated in tissue regeneration in wound healing [8].
mechanism like histone deacetylation has been proposed as other negative regulators of PDPN expression [22].
Regulators of PDPN expression
Role of PDPN in cancer initiation, progression, and metastasis
A number of physiologic and pathologic factors regulate PDPN expression. Normal differentiation factors, such as Prox-1 and IL-3, critical regulators of LEC differentiation, are found to be involved in controlling PDPN transcription [18, 19]. Downstream molecules of different cell signaling pathways like transcription factors AP-1 and proinflammatory cytokines like IL-1β, IL-6, IL-22, TNF-α, TGF-β, TGF-β1, or IFN-γ are observed to be associated with upregulated PDPN expression [8, 20]. In inflammatory conditions like rheumatoid arthritis, PDPN expression was observed to be enhanced by proinflammatory cytokines like IL-1β, TNFα, and TGF-β1 [21]. PDPN expression was also induced by epidermal growth factor, basic fibroblast growth factor in the MCF7 breast cancer cell line, and by bradykinin in 3T3 fibroblasts, by interleukin-3 in dermal LECs and by transforming growth factor beta in human fibrosarcoma cells [12]. Ohta et al. proposed that TGF-β receptor/Smad signaling pathway is directly involved in the positive regulation of PDPN expression in oral squamous cell carcinoma (OSCC) cell lines as they observed the phosphorylation and localization of Smad2 into the nucleus in PDPN positive tumor cells and neighboring stromal cells (cancerassociated fibroblasts). These TGF-β receptor/Smad signaling pathway-activated cancer-associated fibroblasts reportedly act as sources for secretion of cytokines such as TGF-β and hepatocyte growth factor (HGF) and hence probably responsible for intratumoral and peritumoral PDPN expression [22]. According to Hantusch et al., PDPN immunoexpression by basal cell layer may be maintained by Smad via regulation of other transcriptional factors like Sp1 and Sp3 as TGF-β receptor/Smad signaling might not alone be sufficient to induce PDPN as stated in some studies [23]. Mounting evidence supports that amongst all cancer microenvironmental growth factors, EGF has the most potential in stimulating PDPN expression via Src-Cas pathway. Src, belonging to one of the 11 nonreceptor tyrosine kinase family, activated by EGF factor lead to subsequent phosphorylation of tyrosine residues on substrates resulting in various cellular responses such as motility, cell division, migration and angiogenesis. Activated Src has been observed to induce PDPN expression through phosphorylation of Cas (crk-associated substrate) and induction of FOS, a component of AP1 transcription factor [24, 25]. PDPN expression is negatively regulated by the squamous differentiation both in vitro and in vivo as Ohta et al. demonstrated its decreased expression in the PDPN-positive cells by enhancing squamous cell differentiation. Epigenetic
Cumulative evidence of constitutive expression of PDPN in various tumor model experiments, both in vitro and in vivo suggests its potential role in carcinogenesis [26, 27]. Atsumi et al. have described PDPN as a marker for tumor initiating cells of squamous cell carcinoma. The PDPN-positive cancer cells exhibited stem-cell-like properties as they had the ability to repopulate and to generate a heterogeneous cancer cell population. Furthermore, these cells showed a higher colony forming efficiency in comparison with negative counterpart. Though the exact role of PDPN in cancer initiation remains elusive, intrinsic mechanism like activation of the SHH signaling pathway by PDPN thought to have contribution in both initiation and progression of tumor [26]. Cancer cell motility is a complex process as both individual and collective migration modes are regulated and complicated by heterologous interaction of tumor cells and microenvironment. Different mechanisms like epithelial mesenchymal transition (EMT), mesenchymal amoeboid transition and collective amoeboid transition which contribute the cancer cell migration and dedifferentiation, largely depends on active remodeling of cell cytoskeleton. Upregulation of PDPN expression was observed to be localized to the membrane extension, i.e., filopodia and lamellopodia in keratinocytes. A conserved motif of three basic residues in cytoplasmic tail of PDPN binds with ERM family of proteins. Overexpression of PDPN induces increased and sustained phosphorylation of ERM proteins, which serves as connectors between integral membrane proteins and actin cytoskeleton. Apart from ERM protein function, the activities of Rho-family GTPases, in particular RhoA, are modulated independently by PDPN. Regulation of RhoA activity is causally involved in the promigratory phenotype observed in PDPN-expressing cancer cells. Decreased stress fibers and increased filopodia formation in PDPN positive cell lead to mesenchymal appearance. These changes, in addition to a downregulation of E-cadherin and other epithelial markers, indicative of cells undergoing EMT were observed [28, 29]. However, Wicki et al. demonstrated that while PDPN overexpression resulted in increased migration and invasion of cancer cells in the absence of a cadherin switch and EMT [30]. Later on, Wicki et al. studied molecular basis underlying the phenomenon of cell invasion induced by PDPN and observed that PDPN does not suppress the cadherin switch or EMT, but is able to mediate an independent pathway of tumor cell invasion [31] (Fig. 1). Recently, CD44, a major hyaluran receptor and also a novel partner for PDPN is linked with directional persistence of motility in
Tumor Biol.
Fig. 1 Podoplanin in tumorogenesis and metastasis: Through EMT pathway—proinflammatory cytokines and growth factors act as positive regulators of PDPN transcription (1). Overexpression of PDPN can induce ERM protein phosphorylation through recruitment of Ezrin moiety followed by its co-localization with cytoplasmic tail of PDPN (2) or through Rho GDP/GTP activation by Rho GTP kinases (3). The consequent effectors Rac-1, Rho-A, and cdc-42 stimulate actin polymerization leading to filopodium formation via Rho/ROCK signaling pathway (4, 5). Together with cadherin switch (E- to N-cadherin), PDPN induces EMT. At the leading edge, glycosylated PDPN promotes directional motility
with co-expression of CD44 (6). PDPN also induces platelet aggregation via CLEC-2 and PLAG domain interaction (inset). Bidirectional induction of tumor cells and activated platelets leads to formation of tumor heteroaggregates (7, 8). Through induction of matrix metaloproteases, PDPN also accelerates the process of metastasis (9). Through non-EMT pathway—cellular events like direct activation of ERM protein by PDPN with downregulation of RhoA, decrease stress fiber formation and coexpression with E-cadherin explains collective cell migration by PDPN in absence of EMT (10, 11)
PDPN-positive epithelial cells. Co-expression of PDPN and CD44, which is dependent on differential glycosylation of both molecules, was found to be upregulated in aggressive cancer cell lines [32]. Platelet aggregation is proposed to be one of the mechanisms influencing tumor metastatic potential by protecting of tumor cells during their transit through the bloodstream, mediate adherence to vascular endothelium, evasion from immunosurveillance and release of various potent bioactive molecules that facilitate tumor cell extravasation and growth at metastatic sites. Various studies support PDPN as a powerful platelet aggregator by binding platelet C-type lectin receptor, CLEC-2 via platelet aggregation stimulating (PLAG) domain which is a highly conserved amino acid sequence in the PDPN extracellular domain. This interaction induces bidirectional signaling causing both clustering PDPN and platelet aggregation which sequentially promoting tumor cell migration and metastasis [33, 34].
stromal elements like cancer-associated fibroblasts (CAFs), provide an essential communication network via secretion of growth factors and chemokines, inducing tumor progression and metastasis. Interestingly, PDPN is now observed to overexpressed by CAFs in different human cancers, i.e., lung adenocarcinomas, colorectal cancer, breast cancer, and lung squamous cell carcinoma [35–37]. Correlation of upregulation of PDPN expression in CAFs with prognosis mainly depends on type of tumor cells and origin of CAFs. In majority of published studies, PDPN expression in CAFs was identified to be a negative predictor for overall patient survival rate. It was also noticed that CAFs isolated from vascular adventitia differentially expressed PDPN gene leading to ectopic expression of PDPN. This consequently exerted elevated RhoA activity making the microenvironment comparatively more conducive for tumor growth than other CAFs [38, 39]. Conversely, in one study of colorectal cancer CAFs found that PDPN expression was correlated with a favorable clinical outcome of patients [40]. This contradictory observation, also supported by in vitro tumor invasion assay, postulated that PDPN-expressing stroma could act as a physical barrier to tumor cell invasion into surrounding stroma, as PDPN being negative charged mucin tend to repel other similar molecule like complement affecting cellular adhesion. However, there
PDPN in tumor microenvironment The tumor microenvironment, the product of a developing crosstalk between different cells types including critical
Tumor Biol. Table 1 Podoplanin expression in different clinical studies of oral cancer and potentially malignant disorder (precancer) Author
Year Oral precancer and cancer
Martin-Villar et al. [50]
2005 Oral cancer and epithelial dysplasia
Yuan et al. [51]
2006
Ohno et al. [57]
2007
Kawaguchi et al. [42] Kreppel et al. [52]
2008 2010
Shi et al. [48]
2010
Shimamura et al. [49]
2011
Funayama et al. [49]
2011
Feng et al. [46] Inoue et al. [24]
2012 2012
Kreppel et al. [45]
2012
Almeida et al. [53]
2013
Observation
Reorganization of the actin cytoskeleton by recruitment of ezrin and the induction of cell-surface protrusions thus inducing tumor cell invasion and migration Oral cancer Podoplanin as predictor of lymph node metastasis and poor clinical outcomes (shortest disease-specific survival) Oral cancer Peritumoral lymphatic vessel density and overexpression in tumor cells indicates podoplanin as a useful biomarker for assessing tumor aggressiveness Oral leukoplakia Independent predictor for oral cancer development Oral cancer Strong association between increased podoplanin expression and cervical lymph node metastasis and poorer 5-year survival rate Oral lichen planus Co-expression of podoplanin and ATP-binding cassette, G2 subfamily (ABCG2) can be used as biomarkers for malignant transformation risk Oral dysplasia and carcinoma in situ Assessment of podoplanin and fascin could improve the diagnostic and grading accuracy for epithelial dysplasia Oral cancer and epithelial dysplasia Overexpression of podoplanin can be correlated to the severity of dysplasia Indicative of carcinogenesis Oral erythroplakia Valuable predictor for evaluation of oral cancer risk Oral leukoplakia, erythroplakia, Role of podoplanin in dysplasia to carcinoma sequence by mediating a intraepithelial neoplasia, and oral signalling pathway through involvement of growth factors like basic cancer fibroblast growth factor, epidermal growth factor, and tumor growth factor-β1 Oral leukoplakia Podoplanin expression may augment the histological grading and can be a biomarker for malignant transformation risk in leukoplakia Oral cancer Predictor for perineural invasion
is insufficient data to fully assess and establish the prognostic value of PDPN expression by CAFs in head and neck cancer [40, 41].
PDPN in oral precancer and cancer Previous studies have already indicated the potential role of this protein in carcinogenesis as overexpression of PDPN in hyperplastic and dysplastic areas [42]. In recent years, studies have reported the overexpression of PDPN in oral premalignacies like oral leukoplakia, oral erythroleukoplakia, carcinoma in situ, and lichen planus [43–48]. These evidences suggest the use of PDPN as a promising biomarker for oral cancer risk in patients with oral premalignancy. Significant contribution in this field was done by Kawaguchi et al. as they reported about the strong association between high PDPN expression and cancer development in oral leukoplakia in a prospective study of 7.5 years of follow-up period [42]. A recent retrospective study conducted by Kreppel et al. also detected similar results if not so impressive as in the previous study [45]. Some authors also suggested the co-expression of PDPN with other markers like ABCG2 (a molecular determinant, for maintaining the side population phenotype in stem cells) or Fascin (an actin-bundling protein) may be better
predictive marker for evaluating oral cancer risk in premalignacies as PDPN expression alone may not be sufficient to promote tumorigenesis because many of the lesions exhibit the protein expression only in the basal layer [46-9]. Though the exact role of PDPN is still debatable, the strong statistical association between PDPN expression and malignant transformation risk support the putative role of this protein in carcinogenesis. Hence, abnormal detection of PDPN-positive cells in the suprabasal epithelial layers significantly correlated with increased oral cancer risk, some authors suggested hypothesis of upward clonal expansion theory during carcinogenesis [42]. Noticeable multilayering expression of PDPN in dysplastic epithelium may indicate the cell migrating activity of PDPN +ve cells within their foci. Subsequent upregulation of matrix metalloproteinases in adjacent extracellular milieu also denotes the ability of invasion to the surrounding either by single or collective cell migration of PDPN expressing cells [47]. Regardless the type of premalignant lesion and nature of study, most authors agreed that validity of histological grading as predictive marker in oral cancer risk assessment is questionable due to substantial interobserver and intraobserver variations. Consistent and significant expression of PDPN in premalignant lesion not only suggest it as a powerful biomarker predicting the true dysplastic lesion by augmentation of established clinical and
Tumor Biol.
histological parameters but also may be helpful to guide treatment selection for surgeons (Table 1). Many studies have revealed altered PDPN expression in various cancers including oral squamous cell carcinoma (OSCC) cells raising a possibility that PDPN may have potential role in not only in carcinogenesis but also in tumor progression, metastasis, and overall prognosis. Martin-Villar et al. first noticed restricted heterogeneous expression of PDPN at the tumor front of OSCC tissue sample with downregulation of E-cadherin inducing redistribution of ezrin to the cell edges, formation of cell-surface protrusions, and reduced Ca2-dependent cell-cell adhesiveness [50]. Further studies were designed to correlate the PDPN expression status with clinical outcome of OSCC patients. Yuan et al. reported significant correlation between high PDPN expression, lymph node metastasis, and diseasespecific survival rate exclusively in oral SCC [51]. A similar study conducted by Kreppel et al. also found strong association between aforementioned variables. The 5-year overall survival (31 %) for the OSCC patients with high levels of PDPN expression was significantly lower than patients with low and moderate expression of PDPN (93 and 65 %, respectively) [52]. In a recent study on expression of podoplanin along with vascular endothelial growth factor-C (VEGF-C) has suggested its prognostic significance in perineural invasion in OSCC, a strong negative predictor of prognosis [53]. Podopanin, being a preferential lymphoendothelial cell marker, has been used to predict tumor metastatic potential by assessment of peritumoral (PLD) and intratumoral lymphatic vascular density (ILD). Various studies report contradictory opinions regarding the association between type of lymphangionesis and prognosis [54-58]. Some investigators suggest a significant association of higher PLD with shorter disease-free survival, while there are studies which also report a correlation between higher PLD and more favorable outcome. Though a significant association between higher PLD and lymph node involvement was observed, there is no evidence showing correlation of PLD with 5-year cumulative survival and disease-free survival [55]. On the contrary, Kyzas et al. and Zhao et al. emphasized ILD to be an independent prognostic factor for mortality [57]. An exclusive study on ILD suggested variability in the distribution of intratumoral lymphangiogenesis within the tumor tissue. Superficial area of tumor tissue showed significantly higher LVD than the deeper areas of tumor near invasion front [58]. Aside from all the controversial results on tumor lymphangiogenesis, PDPN can be used as a specific marker to predict lymphatic dissemination, therapeutic strategy and prognosis in OSCC [54-7].
Podoplanin—a potential target in cancer therapeutics Significant contribution of PDPN in different steps of carcinogenesis has drawn attention of researchers to reveal its
perspectives as a molecular target in cancer therapeutics. Recent experimental and clinical studies have demonstrated the following observations providing a platform to introduce podoplanin as a potential molecular therapeutic target [59–61]. & &
&
Modification in glycosylation of extracellular domain of PDPN by lectins may inhibit tumor cell growth and motility [59]. Suppression of PDPN-induced platelet aggregation by targeted immunotherapeutic agents such as NZ-1 and chimeric antibodies (MS-1), subsequently affecting the tumor progression and metastasis [60, 61]. Extracelluar domain of PDPN can be targeted by recombinant fusion protein to inhibit tumor lymphangiogenesis [62].
Conclusion PDPN, a critical molecule in the signaling pathway involved in development of epithelial dysplasia–carcinoma sequence, can act as a reliable predictor in assessing risk of cancer development in potential malignant disorders of oral cavity. Having a well-documented role in inducing cancer cell migration and metastasis along with its specificity in evaluating lymphovascular invasion, PDPN can be proposed as a novel diagnostic and prognostic biomarker for head and neck squamous cell carcinoma.
Author’s contribution All authors have contributed equally in preparation of the manuscript. Conflicts of interest None Role of funding source None Ethics committee approval Not applicable
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53. dos Santos AA, Oliveira DT, Pereira MC, Faustino SE, Nonogaki S, Carvalho AL, et al. Podoplanin and VEGF-C immunoexpression in oral squamous cell carcinomas: prognostic significance. Anticancer Res. 2013;33(9):3969–76. 54. Zhao D, Pan J, Li XQ, Wang XY, Tang C, Xuan M. Intratumoral lymphangiogenesis in oral squamous cell carcinoma and its clinicopathological significance. J Oral Pathol Med. 2008;37(10):616–25. 55. de Sousa SF, Gleber-Netto FO, de Oliveira-Neto HH, Batista AC, Nogueira Guimarães Abreu MH, de Aguiar MC. Lymphangiogenesis and podoplanin expression in oral squamous cell carcinoma and the associated lymph nodes. Appl Immunohistochem Mol Morphol. 2012;20(6):588–94. 56. Muñoz-Guerra MF, Marazuela EG, Martín-Villar E, Quintanilla M, Gamallo C. Prognostic significance of intratumoral lymphangiogenesis in squamous cell carcinoma of the oral cavity. Cancer. 2004;100(3):553–60. 57. Kyzas PA, Geleff S, Batistatou A, Agnantis NJ, Stefanou D. Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma. J Pathol. 2005;206(2):170– 7. 58. Ohno F, Nakanishi H, Abe A, Seki Y, Kinoshita A, Hasegawa Y, et al. Regional difference in intratumoral lymphangiogenesis of oral squamous cell carcinomas evaluated by immunohistochemistry using D240 and podoplanin antibody: an analysis in comparison with angiogenesis. J Oral Pathol Med. 2007;36(5):281–9. 59. Ochoa-Alvarez JA, Krishnan H, Shen Y, Acharya NK, Han M, et al. Plant lectin can target receptors containing sialic acid, exemplified by podoplanin, to inhibit transformed cell growth and migration. PLoS ONE. 2012;7(7):e41845. doi:10.1371/journal.pone.0041845. 60. Chandramohan V, Bao X, Kato KM, Kato Y, Keir ST, Szafranski SE, et al. Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors. Int J Cancer. 2013;132(10): 2339–48. 61. Takagi S, Sato S, Oh-hara T, Takami M, Koike S, et al. Platelets promote tumor growth and metastasis via direct interaction between Aggrus/podoplanin and CLEC-2. PLoS ONE. 2013;8(8):e73609. doi:10.1371/journal.pone.0073609. 62. Cueni LN et al. Podoplanin-Fc reduces lymphatic vessel formation in vitro and in vivo and causes disseminated intravascular coagulation when transgenically expressed in the skin. Blood. 2010;116(20): 4376–84.
REVIEW
Podoplanin—a novel marker in oral carcinogenesis Niharika Swain & Shwetha V. Kumar & Samapika Routray & Jigna Pathak & Shilpa Patel
Received: 19 April 2014 / Accepted: 19 June 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Podoplanin, a transmembrane sialoglycoprotein, is a specific marker for lymphatic endothelial cells which in recent years has gained prominent notoriety for its role in tumor progression and metastasis. It is an extensively studied biomarker for predictive assessment of malignant transformation as well as biologic behavior in both human precancer and cancer, respectively. This review summarizes the association of podoplanin overexpression in oral potentially malignant disorders and oral cancer with special emphasis on its putative role in carcinogenesis as well as its prospective use in targeted therapy. Keywords Podoplanin . Biomarker . Carcinogenesis . Oral potentially malignant disorder . Oral cancer . Epithelial mesenchymal transition
Introduction Podoplanin, though initially detected in murine osteosarcoma cell lines and lymphatic endothelial cells (LECs), was so coined due to its peculiar expression in podocytes or foot processes of renal corpuscles and its potential influence in maintaining the unique shape of podocytes [1]. Subsequently, several PDPN like homologous proteins such as, the oncofetal antigen M2A recognized by the D2-40 antibody and the type I alveolar cell marker hT1α-2 and gp36, a sialoglycoptrotein of N. Swain (*) : S. V. Kumar : J. Pathak : S. Patel Department of Oral Pathology, MGM Dental College and Hospital, 304, Siddhi Vinayak Residency Sector 9, Plot no. 40 Kamothe, Navi Mumbai 410209, Maharashtra, India e-mail: [email protected] S. Routray Department of Oral Pathology, Institute of Dental Sciences, Siksha ‘O’ Anusandhan University, Bhubaneswar, Odisha, India
vascular endothelium and alveolar epithelium, were indentified in human body [2]. PDPN primarily belongs to the family of type 1 transmembrane sialomucin-like glycoproteins. It consists of 162 amino acids, with an extracellular domain rich in serine and threonine residues, a single hydrophobic membrane spanning domain, and a short cytoplasmic endodomain. The extracellular domain is composed of repetitive mucin sequences with extensive O-glycosylated Ser and Thr residues ensuing in an extended rigid structure and a net negative charge. The extremely short cytoplasmic domain composed of only nine amino acids having a functional motif, i.e., Ser (Ser 167), which is a potential cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylation site and a cluster of highly conserved basic amino acids, recruiting site for proteins of the ezrin, radixin, and moesin (ERM) family [3, 4]. Apart from being a prominent lymphatic endothelial cell marker, PDPN has been reported to have a wide cellular distribution such as osteocytes, osteoblasts [5], odontoblasts [6], enamel epithelia, mesothelial cells [7], epidermal basal layer cells [8], salivary myoepithelial cells [9], choroid plexus epithelial cells [10], thymus type 1 epithelial cells [11], prostate myofibroblasts [12], follicular dendritic cells [13], immature cells like fetal germ cells, and developing Sertoli cells [2, 14]. Physiologically, PDPN seems to have multifaceted role in embryogenic development and growth of various organs like lymphatic system, lungs, and heart in humans. In vasculogenesis, PDPN has a crucial role in the separation of lymphatic from the blood circulatory system. The interaction of platelet and PDPN on lymphatic endothelial cells induces platelet aggregation and prevents blood from flowing into new lymphatic vessels budding from the cardinal vein. Furthermore, continued expression of PDPN into adulthood reinforces its importance in maintaining proper lymphatic architecture. In embryogenesis of the heart and lungs, the tissuespecific intrinsic role of PDPN in cell differentiation and
Tumor Biol.
proliferation is speculated in various studies [15–17]. In addition to its physiological role, PDPN has also been implicated in tissue regeneration in wound healing [8].
mechanism like histone deacetylation has been proposed as other negative regulators of PDPN expression [22].
Regulators of PDPN expression
Role of PDPN in cancer initiation, progression, and metastasis
A number of physiologic and pathologic factors regulate PDPN expression. Normal differentiation factors, such as Prox-1 and IL-3, critical regulators of LEC differentiation, are found to be involved in controlling PDPN transcription [18, 19]. Downstream molecules of different cell signaling pathways like transcription factors AP-1 and proinflammatory cytokines like IL-1β, IL-6, IL-22, TNF-α, TGF-β, TGF-β1, or IFN-γ are observed to be associated with upregulated PDPN expression [8, 20]. In inflammatory conditions like rheumatoid arthritis, PDPN expression was observed to be enhanced by proinflammatory cytokines like IL-1β, TNFα, and TGF-β1 [21]. PDPN expression was also induced by epidermal growth factor, basic fibroblast growth factor in the MCF7 breast cancer cell line, and by bradykinin in 3T3 fibroblasts, by interleukin-3 in dermal LECs and by transforming growth factor beta in human fibrosarcoma cells [12]. Ohta et al. proposed that TGF-β receptor/Smad signaling pathway is directly involved in the positive regulation of PDPN expression in oral squamous cell carcinoma (OSCC) cell lines as they observed the phosphorylation and localization of Smad2 into the nucleus in PDPN positive tumor cells and neighboring stromal cells (cancerassociated fibroblasts). These TGF-β receptor/Smad signaling pathway-activated cancer-associated fibroblasts reportedly act as sources for secretion of cytokines such as TGF-β and hepatocyte growth factor (HGF) and hence probably responsible for intratumoral and peritumoral PDPN expression [22]. According to Hantusch et al., PDPN immunoexpression by basal cell layer may be maintained by Smad via regulation of other transcriptional factors like Sp1 and Sp3 as TGF-β receptor/Smad signaling might not alone be sufficient to induce PDPN as stated in some studies [23]. Mounting evidence supports that amongst all cancer microenvironmental growth factors, EGF has the most potential in stimulating PDPN expression via Src-Cas pathway. Src, belonging to one of the 11 nonreceptor tyrosine kinase family, activated by EGF factor lead to subsequent phosphorylation of tyrosine residues on substrates resulting in various cellular responses such as motility, cell division, migration and angiogenesis. Activated Src has been observed to induce PDPN expression through phosphorylation of Cas (crk-associated substrate) and induction of FOS, a component of AP1 transcription factor [24, 25]. PDPN expression is negatively regulated by the squamous differentiation both in vitro and in vivo as Ohta et al. demonstrated its decreased expression in the PDPN-positive cells by enhancing squamous cell differentiation. Epigenetic
Cumulative evidence of constitutive expression of PDPN in various tumor model experiments, both in vitro and in vivo suggests its potential role in carcinogenesis [26, 27]. Atsumi et al. have described PDPN as a marker for tumor initiating cells of squamous cell carcinoma. The PDPN-positive cancer cells exhibited stem-cell-like properties as they had the ability to repopulate and to generate a heterogeneous cancer cell population. Furthermore, these cells showed a higher colony forming efficiency in comparison with negative counterpart. Though the exact role of PDPN in cancer initiation remains elusive, intrinsic mechanism like activation of the SHH signaling pathway by PDPN thought to have contribution in both initiation and progression of tumor [26]. Cancer cell motility is a complex process as both individual and collective migration modes are regulated and complicated by heterologous interaction of tumor cells and microenvironment. Different mechanisms like epithelial mesenchymal transition (EMT), mesenchymal amoeboid transition and collective amoeboid transition which contribute the cancer cell migration and dedifferentiation, largely depends on active remodeling of cell cytoskeleton. Upregulation of PDPN expression was observed to be localized to the membrane extension, i.e., filopodia and lamellopodia in keratinocytes. A conserved motif of three basic residues in cytoplasmic tail of PDPN binds with ERM family of proteins. Overexpression of PDPN induces increased and sustained phosphorylation of ERM proteins, which serves as connectors between integral membrane proteins and actin cytoskeleton. Apart from ERM protein function, the activities of Rho-family GTPases, in particular RhoA, are modulated independently by PDPN. Regulation of RhoA activity is causally involved in the promigratory phenotype observed in PDPN-expressing cancer cells. Decreased stress fibers and increased filopodia formation in PDPN positive cell lead to mesenchymal appearance. These changes, in addition to a downregulation of E-cadherin and other epithelial markers, indicative of cells undergoing EMT were observed [28, 29]. However, Wicki et al. demonstrated that while PDPN overexpression resulted in increased migration and invasion of cancer cells in the absence of a cadherin switch and EMT [30]. Later on, Wicki et al. studied molecular basis underlying the phenomenon of cell invasion induced by PDPN and observed that PDPN does not suppress the cadherin switch or EMT, but is able to mediate an independent pathway of tumor cell invasion [31] (Fig. 1). Recently, CD44, a major hyaluran receptor and also a novel partner for PDPN is linked with directional persistence of motility in
Tumor Biol.
Fig. 1 Podoplanin in tumorogenesis and metastasis: Through EMT pathway—proinflammatory cytokines and growth factors act as positive regulators of PDPN transcription (1). Overexpression of PDPN can induce ERM protein phosphorylation through recruitment of Ezrin moiety followed by its co-localization with cytoplasmic tail of PDPN (2) or through Rho GDP/GTP activation by Rho GTP kinases (3). The consequent effectors Rac-1, Rho-A, and cdc-42 stimulate actin polymerization leading to filopodium formation via Rho/ROCK signaling pathway (4, 5). Together with cadherin switch (E- to N-cadherin), PDPN induces EMT. At the leading edge, glycosylated PDPN promotes directional motility
with co-expression of CD44 (6). PDPN also induces platelet aggregation via CLEC-2 and PLAG domain interaction (inset). Bidirectional induction of tumor cells and activated platelets leads to formation of tumor heteroaggregates (7, 8). Through induction of matrix metaloproteases, PDPN also accelerates the process of metastasis (9). Through non-EMT pathway—cellular events like direct activation of ERM protein by PDPN with downregulation of RhoA, decrease stress fiber formation and coexpression with E-cadherin explains collective cell migration by PDPN in absence of EMT (10, 11)
PDPN-positive epithelial cells. Co-expression of PDPN and CD44, which is dependent on differential glycosylation of both molecules, was found to be upregulated in aggressive cancer cell lines [32]. Platelet aggregation is proposed to be one of the mechanisms influencing tumor metastatic potential by protecting of tumor cells during their transit through the bloodstream, mediate adherence to vascular endothelium, evasion from immunosurveillance and release of various potent bioactive molecules that facilitate tumor cell extravasation and growth at metastatic sites. Various studies support PDPN as a powerful platelet aggregator by binding platelet C-type lectin receptor, CLEC-2 via platelet aggregation stimulating (PLAG) domain which is a highly conserved amino acid sequence in the PDPN extracellular domain. This interaction induces bidirectional signaling causing both clustering PDPN and platelet aggregation which sequentially promoting tumor cell migration and metastasis [33, 34].
stromal elements like cancer-associated fibroblasts (CAFs), provide an essential communication network via secretion of growth factors and chemokines, inducing tumor progression and metastasis. Interestingly, PDPN is now observed to overexpressed by CAFs in different human cancers, i.e., lung adenocarcinomas, colorectal cancer, breast cancer, and lung squamous cell carcinoma [35–37]. Correlation of upregulation of PDPN expression in CAFs with prognosis mainly depends on type of tumor cells and origin of CAFs. In majority of published studies, PDPN expression in CAFs was identified to be a negative predictor for overall patient survival rate. It was also noticed that CAFs isolated from vascular adventitia differentially expressed PDPN gene leading to ectopic expression of PDPN. This consequently exerted elevated RhoA activity making the microenvironment comparatively more conducive for tumor growth than other CAFs [38, 39]. Conversely, in one study of colorectal cancer CAFs found that PDPN expression was correlated with a favorable clinical outcome of patients [40]. This contradictory observation, also supported by in vitro tumor invasion assay, postulated that PDPN-expressing stroma could act as a physical barrier to tumor cell invasion into surrounding stroma, as PDPN being negative charged mucin tend to repel other similar molecule like complement affecting cellular adhesion. However, there
PDPN in tumor microenvironment The tumor microenvironment, the product of a developing crosstalk between different cells types including critical
Tumor Biol. Table 1 Podoplanin expression in different clinical studies of oral cancer and potentially malignant disorder (precancer) Author
Year Oral precancer and cancer
Martin-Villar et al. [50]
2005 Oral cancer and epithelial dysplasia
Yuan et al. [51]
2006
Ohno et al. [57]
2007
Kawaguchi et al. [42] Kreppel et al. [52]
2008 2010
Shi et al. [48]
2010
Shimamura et al. [49]
2011
Funayama et al. [49]
2011
Feng et al. [46] Inoue et al. [24]
2012 2012
Kreppel et al. [45]
2012
Almeida et al. [53]
2013
Observation
Reorganization of the actin cytoskeleton by recruitment of ezrin and the induction of cell-surface protrusions thus inducing tumor cell invasion and migration Oral cancer Podoplanin as predictor of lymph node metastasis and poor clinical outcomes (shortest disease-specific survival) Oral cancer Peritumoral lymphatic vessel density and overexpression in tumor cells indicates podoplanin as a useful biomarker for assessing tumor aggressiveness Oral leukoplakia Independent predictor for oral cancer development Oral cancer Strong association between increased podoplanin expression and cervical lymph node metastasis and poorer 5-year survival rate Oral lichen planus Co-expression of podoplanin and ATP-binding cassette, G2 subfamily (ABCG2) can be used as biomarkers for malignant transformation risk Oral dysplasia and carcinoma in situ Assessment of podoplanin and fascin could improve the diagnostic and grading accuracy for epithelial dysplasia Oral cancer and epithelial dysplasia Overexpression of podoplanin can be correlated to the severity of dysplasia Indicative of carcinogenesis Oral erythroplakia Valuable predictor for evaluation of oral cancer risk Oral leukoplakia, erythroplakia, Role of podoplanin in dysplasia to carcinoma sequence by mediating a intraepithelial neoplasia, and oral signalling pathway through involvement of growth factors like basic cancer fibroblast growth factor, epidermal growth factor, and tumor growth factor-β1 Oral leukoplakia Podoplanin expression may augment the histological grading and can be a biomarker for malignant transformation risk in leukoplakia Oral cancer Predictor for perineural invasion
is insufficient data to fully assess and establish the prognostic value of PDPN expression by CAFs in head and neck cancer [40, 41].
PDPN in oral precancer and cancer Previous studies have already indicated the potential role of this protein in carcinogenesis as overexpression of PDPN in hyperplastic and dysplastic areas [42]. In recent years, studies have reported the overexpression of PDPN in oral premalignacies like oral leukoplakia, oral erythroleukoplakia, carcinoma in situ, and lichen planus [43–48]. These evidences suggest the use of PDPN as a promising biomarker for oral cancer risk in patients with oral premalignancy. Significant contribution in this field was done by Kawaguchi et al. as they reported about the strong association between high PDPN expression and cancer development in oral leukoplakia in a prospective study of 7.5 years of follow-up period [42]. A recent retrospective study conducted by Kreppel et al. also detected similar results if not so impressive as in the previous study [45]. Some authors also suggested the co-expression of PDPN with other markers like ABCG2 (a molecular determinant, for maintaining the side population phenotype in stem cells) or Fascin (an actin-bundling protein) may be better
predictive marker for evaluating oral cancer risk in premalignacies as PDPN expression alone may not be sufficient to promote tumorigenesis because many of the lesions exhibit the protein expression only in the basal layer [46-9]. Though the exact role of PDPN is still debatable, the strong statistical association between PDPN expression and malignant transformation risk support the putative role of this protein in carcinogenesis. Hence, abnormal detection of PDPN-positive cells in the suprabasal epithelial layers significantly correlated with increased oral cancer risk, some authors suggested hypothesis of upward clonal expansion theory during carcinogenesis [42]. Noticeable multilayering expression of PDPN in dysplastic epithelium may indicate the cell migrating activity of PDPN +ve cells within their foci. Subsequent upregulation of matrix metalloproteinases in adjacent extracellular milieu also denotes the ability of invasion to the surrounding either by single or collective cell migration of PDPN expressing cells [47]. Regardless the type of premalignant lesion and nature of study, most authors agreed that validity of histological grading as predictive marker in oral cancer risk assessment is questionable due to substantial interobserver and intraobserver variations. Consistent and significant expression of PDPN in premalignant lesion not only suggest it as a powerful biomarker predicting the true dysplastic lesion by augmentation of established clinical and
Tumor Biol.
histological parameters but also may be helpful to guide treatment selection for surgeons (Table 1). Many studies have revealed altered PDPN expression in various cancers including oral squamous cell carcinoma (OSCC) cells raising a possibility that PDPN may have potential role in not only in carcinogenesis but also in tumor progression, metastasis, and overall prognosis. Martin-Villar et al. first noticed restricted heterogeneous expression of PDPN at the tumor front of OSCC tissue sample with downregulation of E-cadherin inducing redistribution of ezrin to the cell edges, formation of cell-surface protrusions, and reduced Ca2-dependent cell-cell adhesiveness [50]. Further studies were designed to correlate the PDPN expression status with clinical outcome of OSCC patients. Yuan et al. reported significant correlation between high PDPN expression, lymph node metastasis, and diseasespecific survival rate exclusively in oral SCC [51]. A similar study conducted by Kreppel et al. also found strong association between aforementioned variables. The 5-year overall survival (31 %) for the OSCC patients with high levels of PDPN expression was significantly lower than patients with low and moderate expression of PDPN (93 and 65 %, respectively) [52]. In a recent study on expression of podoplanin along with vascular endothelial growth factor-C (VEGF-C) has suggested its prognostic significance in perineural invasion in OSCC, a strong negative predictor of prognosis [53]. Podopanin, being a preferential lymphoendothelial cell marker, has been used to predict tumor metastatic potential by assessment of peritumoral (PLD) and intratumoral lymphatic vascular density (ILD). Various studies report contradictory opinions regarding the association between type of lymphangionesis and prognosis [54-58]. Some investigators suggest a significant association of higher PLD with shorter disease-free survival, while there are studies which also report a correlation between higher PLD and more favorable outcome. Though a significant association between higher PLD and lymph node involvement was observed, there is no evidence showing correlation of PLD with 5-year cumulative survival and disease-free survival [55]. On the contrary, Kyzas et al. and Zhao et al. emphasized ILD to be an independent prognostic factor for mortality [57]. An exclusive study on ILD suggested variability in the distribution of intratumoral lymphangiogenesis within the tumor tissue. Superficial area of tumor tissue showed significantly higher LVD than the deeper areas of tumor near invasion front [58]. Aside from all the controversial results on tumor lymphangiogenesis, PDPN can be used as a specific marker to predict lymphatic dissemination, therapeutic strategy and prognosis in OSCC [54-7].
Podoplanin—a potential target in cancer therapeutics Significant contribution of PDPN in different steps of carcinogenesis has drawn attention of researchers to reveal its
perspectives as a molecular target in cancer therapeutics. Recent experimental and clinical studies have demonstrated the following observations providing a platform to introduce podoplanin as a potential molecular therapeutic target [59–61]. & &
&
Modification in glycosylation of extracellular domain of PDPN by lectins may inhibit tumor cell growth and motility [59]. Suppression of PDPN-induced platelet aggregation by targeted immunotherapeutic agents such as NZ-1 and chimeric antibodies (MS-1), subsequently affecting the tumor progression and metastasis [60, 61]. Extracelluar domain of PDPN can be targeted by recombinant fusion protein to inhibit tumor lymphangiogenesis [62].
Conclusion PDPN, a critical molecule in the signaling pathway involved in development of epithelial dysplasia–carcinoma sequence, can act as a reliable predictor in assessing risk of cancer development in potential malignant disorders of oral cavity. Having a well-documented role in inducing cancer cell migration and metastasis along with its specificity in evaluating lymphovascular invasion, PDPN can be proposed as a novel diagnostic and prognostic biomarker for head and neck squamous cell carcinoma.
Author’s contribution All authors have contributed equally in preparation of the manuscript. Conflicts of interest None Role of funding source None Ethics committee approval Not applicable
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