Podocalyxin in the Diagnosis and Treatment of Cancer *

Although their functions in tumour development and progression are not well understood, several secreted and membrane-associated mucins have proven to be valuable diagnostic and prognostic markers in human cancer and many of these are targets for cancer vaccines and therapies (Singh et al., 2008; Chauhan et al., 2009; Kufe, 2009). Podocalyxin-like protein 1 (podocalyxin, gene name PODXL) is a member of the CD34 family of sialomucins. Podocalyxin (PC), also called PCLP1, gp135, MEP21, and thrombomucin, is a single-pass type I transmembrane protein primarily expressed by vascular endothelia, specialized kidney epithelial cells called podocytes, and a limited set of hematopoietic progenitor cells in adult mice and humans (Nielsen & McNagny, 2009b). Expression of PC has also been noted on the luminal face of kidney tubule cells, breast and ductal lumens, oviductal luminal cells, mesothelial cells and neurons in normal mammalian tissues. Polymorphisms and inappropriate or increased expression of PC is linked to several human cancers including germ-line cancers, several carcinomas, malignant astrocytoma and leukemia. In many of these cancers, detection of high levels of PC expression is associated with highgrade, aggressive tumours, increased risk of metastases and poor prognosis. In this review, we will examine the known and proposed functions of PC in normal (adult and embryonic) and cancerous cells, the molecular mechanisms regulating these functions, and potential applications of PC as a molecular marker in the diagnosis, prognosis and treatment of cancer.


Introduction
Although their functions in tumour development and progression are not well understood, several secreted and membrane-associated mucins have proven to be valuable diagnostic and prognostic markers in human cancer and many of these are targets for cancer vaccines and therapies (Singh et al., 2008;Chauhan et al., 2009;Kufe, 2009).Podocalyxin-like protein 1 (podocalyxin, gene name PODXL) is a member of the CD34 family of sialomucins.Podocalyxin (PC), also called PCLP1, gp135, MEP21, and thrombomucin, is a single-pass type I transmembrane protein primarily expressed by vascular endothelia, specialized kidney epithelial cells called podocytes, and a limited set of hematopoietic progenitor cells in adult mice and humans (Nielsen & McNagny, 2009b).Expression of PC has also been noted on the luminal face of kidney tubule cells, breast and ductal lumens, oviductal luminal cells, mesothelial cells and neurons in normal mammalian tissues.Polymorphisms and inappropriate or increased expression of PC is linked to several human cancers including germ-line cancers, several carcinomas, malignant astrocytoma and leukemia.In many of these cancers, detection of high levels of PC expression is associated with highgrade, aggressive tumours, increased risk of metastases and poor prognosis.In this review, we will examine the known and proposed functions of PC in normal (adult and embryonic) and cancerous cells, the molecular mechanisms regulating these functions, and potential applications of PC as a molecular marker in the diagnosis, prognosis and treatment of cancer.

Podocalyxin expression and normal function in mammals 2.1 Podocalyxin has an essential role in the development and function of the kidney glomerulus
PC is named for its expression on specialized kidney epithelial cells called podocytes.On these cells, PC is required for the development of the foot process that, together with fenestrated vascular endothelia, form the filtration apparatus of the glomerulus (reviewed in Nielsen & McNagny, 2009b).Unlike vascular and hematopoietic tissues where other members of the CD34 family are co-expressed, PC is the only CD34-family sialomucin expressed on podocytes and therefore serves a non-redundant function in podocyte development.Germ-line deletion of Podxl in mice results in anuria and death within 24 hours of birth due to the failure of embryonic podocytes to undergo appropriate morphogenesis and form foot processes -one of the most potent mucin-dependent knockout phenotypes described (Doyonnas et al., 2001).Although many tissue types and cell lineages express PC during mouse embryogenesis (and presumably human), PC's only essential function identified so far is the formation of the glomerular filtration apparatus of the kidney.However, we are beginning to discover more subtle roles for PC in the development, morphogenesis, polarization and motility of cells that form the lumen of tubules and ducts (or boundary elements in tissues) including kidney tubules, ovary ducts, mammary ducts and vascular lumens.For example, germline deletion of Podxl in mice causes a delay in the formation of lumens between adjacent endothelial cells of the developing aorta (Strilic et al., 2009) and reduces the adhesion of the mesothelia lining of the gut surface mesothelia to the umbilicus during embryogenesis (Doyonnas et al., 2001).From these and other embryonic analyses we have developed the concept that podocalyxin acts a general anti-adhesive that aids in the demarcation of tissue boundaries (McNagny et al., 1997;Doyonnas et al., 2001).PC expression is also required for efficient neurite outgrowth and branching in the developing brain (Vitureira et al., 2005(Vitureira et al., , 2010)).Despite these subtle developmental delays or abnormalities, in all tissues we have examined so far, save the kidney, Podxl -/-mouse neonates appear to be remarkably normal.
In adult mice and humans, PC is primarily restricted to vascular endothelia, kidney podocytes and a subset of hematopoietic stem cells.In addition, PC expression has also been reported on some mature blood types including mouse "stress" erythroid progenitors (and anemia-induced erythroblasts) (Doyonnas et al., 2005;Sathyanarayana et al., 2007;Maltby et al., 2009) and activated rat platelets (Miettinen et al., 1999).A l t h o u g h o t h e r s a n d w e h a v e p r o p o s e d a r o l e f o r P C ( a n d t h e C D 3 4 a n t i g e n ) i n facilitating the trafficking of hematopoietic progenitors cells to the bone marrow (BM) as well as more mature blood cells from the BM or periphery to sites of inflammation (Nielsen & McNagny, 2008, 2009a, 2009b), the function of PC expression on normal hematopoietic cells remains enigmatic.In summary, although PC has a critical role in kidney development, PC appears to be dispensable for the normal development of other tissues we have examined including vascular endothelia, breast duct epithelia and hematopoietic tissues.However, after increasingly more detailed analyses, we have also found that PC has subtle roles in these tissues under conditions of tissue remodeling and development that may have consequences in disease pathogenesis (unpublished observations).

Transcriptional regulation of PODXL expression
The regulation of PODXL expression is modulated by several transcription factors, epigenetic methylation of CpG islands in the PODXL promoter and by expression of micro RNA (miR-199a2).Depending on the cell lineage and disease state, PODXL expression can be induced by Sp1 (specificity protein 1) and Wilms' tumour suppressor protein 1 (WT1).The PODXL promoter contains multiple binding sites for Sp1 and there is evidence of direct binding of WT1 to the PODXL promoter (Palmer et al., 2001;Butta et al., 2006).While Sp1 is expressed in many cell types and has many targets in the human genome, Sp1 is upregulated in many cancers including epithelial carcinomas (reviewed in Li & Davie, 2010).WT1 is primarily expressed in developing kidney and in the podocytes of the glomerulus in normal kidney.However, WT1 is overexpressed in cancers of the kidney, lung and breast and in acute leukemia and myelodysplastic syndromes.Thus, both Sp1 and WT1 may influence the expression of PODXL in cancers.Adult erythroid cells express PC in response to anemic stress via erythropoietin receptor (EpoR)-dependent activation of the signal transducer and activator of transcription 5 (STAT5) (Sathyanarayana et al., 2007).Although PODXL expression via STAT5 has not been demonstrated in other cell types, STAT5 activation can be induced in epithelial cells by a variety of mechanisms.For example, with respect to the pathogenesis of carcinomas (especially prostate and breast cancers) STAT5 activation downstream of the prolactin receptor activation is one possible route to enhanced PC expression (Jacobson et al., 2010).Transcriptional repressors of PODXL include the ubiquitously expressed tumour suppressor transcription factor p53 (TP53) and the integrin-associated adaptor protein PINCH1 (LIMS1) (Stanhope-Baker et al., 2004;Wang et al., 2011).PINCH1 is normally associated with focal adhesion complexes in podocytes but becomes dissociated from these in response to transforming growth factor beta (TGF) stimulation (Wang et al., 2011).PINCH1 then enters the nucleus and antagonizes WT1 transcriptional activation of PODXL (Wang et al., 2011).Since TGF can induce PODXL expression in a human lung adenocarcinoma cell line (see §3.5.1) (Meng et al., 2011), the role of TGF and PINCH1 in the regulation of PODXL expression may be specific to podocytes (or normal, adherent epithelia).Finally, PODXL is a target for miR-199a2, a micro RNA that is normally expressed in epithelial cells of many tissues (Cheung et al., 2011).miR-199a2 targets (represses) a collection of transcripts and has a putative role as a tumour suppressor in several human cancers (see §3.7.2).

Podocalyxin is a diagnostic marker and prognostic indicator in cancer
Within the last 5-7 years, retrospective analysis of tumour tissue archives by histology, protein and transcript expression analyses have identified increased PC expression or PODXL gene polymorphisms in several human cancers.In this section, we will outline what is known of PC expression patterns in human cancers including epithelial carcinomas (kidney, breast, thyroid, lung, ovarian, prostate and gastrointestinal cancer), germ-cell tumours (testicular cancer), astrocytomas (brain cancer) and leukemia (Table 1).In general, in studies where PC expression has been correlated with tumour behaviour and patient outcome data, PC expression in primary tumour cells is associated with increased tumour aggressiveness, risk of distant metastases and poor prognosis.Mechanistic studies using human cancer cell lines suggest that PC expression in tumour cells is not just merely correlative with aggressiveness, but may have a direct contribution to tumour progression, survival and metastases.In addition to the potential this wealth of data offers for the development of PC-targeted adjuvant treatments for systemic cancer, these findings and accompanying mechanistic studies provide insights into the molecular mechanisms of tumour progression and metastases.While it is clear that there remain many technical challenges for the development of PC-targeted cancer treatments, implementation of clinically beneficial diagnostics is feasible and compatible with existing technology and methods, requiring only a concerted validation effort.

Podocalyxin expression profile
Diagnostic & Prognostic Significance §3.1.1Kidney (Renal cell carcinoma)  PC protein expression 4-fold higher in cancerous cells of rare subset of RCC tumours (~10%)  PC expression in primary RCC correlates with risk of metastasis and poor patient outcome.Hazard ratios (HR) (PC + vs. PC -RCC primary tumors) HR = 3.6 (metastasis-free survival) HR = 7.5 (disease-specific survival) (Hsu et al., 2010)  PC expression in tumor cells correlates with increased risk of aggressive tumor phenotype, and is an independent predictor of distant metastases and poor prognosis §3.1.2Kidney (Nephroblastoma)  Higher PC transcript expression ( 1.5 fold) in more aggressive anaplastic (advanced grade) compared to low grade nephroblastoma (Stanhope-Baker et al., 2004)  Diagnostic marker (transcript) of anaplastic grade nephroblastoma §3.2 Breast carcinoma  High PC protein expression in rare "node-negative" subset of breast carcinoma (~6%)  PC overexpression in primary breast tumours associated with increased relative risk of poor outcome (RR = 8.45) (Somasiri et al., 2004)  PC expression in tumor cells correlates with increased risk of aggressive tumor phenotype, and is an independent predictor of distant metastases and poor prognosis §3.3 Ovarian carcinoma  High expression of PC on tumour cells in 67% of all ovarian epithelial tumours and 87% of high-grade serous epithelial ovarian carcinoma  PC cytoplasmic expression not associated with diseaseoutcome in high-grade serous ovarian tumours, but cell surface expression of PC is an independent predictor of poor patient outcome with HR ~ 1.6 (compared to PCnegative high-grade serous ovarian tumor) (Cipollone et al, submitted)  Prognostic marker of poor outcome in high-grade serous ovarian cancer when PC overexpression is cell surface localized §3.4 Thyroid carcinoma  High expression of PC protein common (~52% cases) in undifferentiated thyroid carcinoma (UTC) subtype but not detected in normal thyroid epithelia or other thyroid cancer cells (Yasuoka et al., 2008)  Diagnostic and staging marker of undifferentiated thyroid carcinoma §3.5 Lung (small-cell lung carcinoma)  PC protein expression detected in tumour cells in majority (~87.5%) in small-cell lung carcinoma (SCLC) (Koch et al., 2008)  Diagnostic marker (tissue biopsy) §3.6 Prostate carcinoma  PODXL germ-line polymorphism and SNP are associated with increased risk of developing prostate cancer with an aggressive tumor.In-frame single or double deletion polymorphism in exon 1 associated with increased risk of prostate cancer Odds ratio (OR) = 2.14-2.58(all prostate cancer) OR = 3.04-4.42(aggressive disease) SNP (G340A) resulting in missense mutation associated with increased risk of prostate cancer OR = 1.48 (all prostate cancer) (Neville et al., 2002)  Possible increased PC expression in metastatic tumor but not primary prostate tumours (Fig. 5) (Yu et al., 2004;Chandran et al., 2007)  Genetic marker of prostate cancer risk  High expression of PC a potential prognostic marker of tumour aggressiveness and metastases

Podocalyxin expression profile
Diagnostic & Prognostic Significance §3.7 Testicular (germ cell tumour)  PC antigens (GCTM-2, TRA-1-60/81) detected in serum of testicular cancer patients (35-75% NSGCT cases) (Schopperle et al., 2003)  PODXL-targeting miR-199a downregulated in testicular cancer (Cheung et al., 2011)  Potential serum marker for testicular cancer  PC potentially equally or more robust marker of testicular cancer compared to  -fetoprotein or chorionic gonadotrophin §3.8.1 Liver (hepatocellular carcinoma (HCC))  High expression of PC protein detected in sinusoidal endothelial associated with HCC but not normal adjacent tissue or cirrhotic lesions (Chen et al., 2004;Heukamp et al., 2006)  Differential diagnosis of HCC vs. cirrhotic lesions (tissue biopsy) §3.8.2 Pancreatic ductal adenocarcinoma  PC protein expressed in primary tumours of 42% pancreatic ductal adenocarcinoma (PDAC) and 30% ampullary ductal carcinoma but rarely in other primary tumours of GI origin (e.g., liver, colon, esophagus)  High PC expression more often detected in high-grade PDAC (53% Grade 3, 48% Grade 2 and 18% Grade 1) (Ney et al., 2007)  Possible identification of primary tissue of metastatic tumours from a suspected GI source §3.8.3 Colon carcinoma  PC protein and peptides detected in media and lysate of colon carcinoma cell lines.
Expression not yet determined in colon cancer primary tumours.(Thomas et al., 2009)  Potential serum marker for colon cancer §3.9 Hematopoietic  PC protein expression commonly detected (77-87% cases) in diffuse pattern (immunohistology) in formalin-fixed bone marrow biopsy in majority cases of AML, ALL and myeloid sarcoma (Kelley et al., 2005). Cell surface expression in 20% AML from fresh blood or bone marrow samples (flow cytometry)  (Riccioni et al., 2006).
 Potential diagnostic marker (tissue biopsy), however significance to disease phenotype and outcome not known  Potential surface marker of AML blasts in peripheral blood §3.10 Brain (astrocytoma)  High PC protein and transcript expression in ~50% cases of high-grade malignant astrocytomas (anaplastic astrocytoma and glioblastoma) but not low-grade astrocytic tumours (Hayatsu et al., 2008).
 Diagnostic marker of high grade/anaplastic astrocytoma Table 1.Summary of podocalyxin expression profiles in human cancers.

Kidney cancer 3.1.1 Renal cell carcinoma
Renal cell carcinoma (RCC), which accounts for 85% of renal cancers, originates from epithelial cells of the renal tubules (Cohen & McGovern, 2005).In a retrospective study, PC protein expression was examined in a collection of 303 formalin-fixed, paraffin-embedded RCC tumours (Hsu et al., 2010).Of these, 29 tumour specimens (9.6%) were found to express PC protein in cancerous cells (4-fold over non-expressors) (Hsu et al., 2010).In contrast, in normal kidney and the majority of RCC patient tumours, PC is only detected in the glomerulus and vascular endothelia (Hsu et al., 2010).Patient outcome data showed that PC-positive RCC tumours were much more likely to be high-grade, advanced-stage tumours (Hsu et al., 2010).Importantly, patients with PC-positive RCC also had decreased disease-specific and metastasis-free survival and PC-positive RCC tumours (stage I-III) were more likely to result in distant metastases (i.e., non-lymph node sites) (Hsu et al., 2010).These findings suggest that PC expression in primary RCC tumours is a strong and independent predictor of distant metastasis and poor prognosis (Hsu et al., 2010).Thus, by identifying a rare, high-risk RCC sub-type, evaluation of PC expression has clear potential diagnostic and prognostic value.

Wilms' tumor (Nephroblastoma)
Wilms' tumour, or nephroblastoma, the most common pediatric kidney cancer, has a unique histological presentation indicative of aberrant or incomplete kidney development during embryogenesis (Huff, 2011).Unlike most of the cancers discussed in this review, PC expression (transcript) is significantly reduced in 64 patient nephroblastoma samples (relative mean 0.29) compared to pooled normal fetal kidney (Stanhope-Baker et al., 2004).Although WT1 is a positive regulator of PODXL expression (Palmer et al., 2001), and loss of WT1 expression is associated with some Wilms' tumours, there was no evidence of a correlation between PODXL and WT1 expression in this study (Stanhope-Baker et al., 2004).However, perhaps of diagnostic value, this same study showed that PODXL expression was increased in more aggressive, anaplastic tumours compared to 40 non-anaplastic tumors (Stanhope-Baker et al., 2004).Notably, functional loss of p53 is associated with the most aggressive, anaplastic nephroblastomas with poor prognosis.As p53 negatively regulates PODXL expression (Stanhope-Baker et al., 2004), enhanced PODXL expression in anaplastic nephroblastomas may be explained directly by this genetic pathway.Although more study is required to know if enhanced PODXL expression in anaplastic nephroblastoma has a direct role in promoting tumour aggressiveness, expression of PODXL in nephroblastoma may be useful as a marker of poorly differentiated, anaplastic tumours.

Breast carcinoma 3.2.1 Expression of podocalyxin is an independent predictor of poor outcome in invasive breast carcinoma
In normal human breast tissue, expression of PC protein is relatively low and restricted to the apical face of the luminal duct epithelia and, as in most tissues, PC also marks the lumen of vascular endothelia in the breast (Somasiri et al., 2004).However, in a survey of tissue microarray constructed from 272 primary tumour samples from patients with locally invasive breast cancer, high expression of PC protein was detected in a small subset (6%) of invasive breast carcinomas (Somasiri et al., 2004).Notably, PC-positive primary tumours were associated with significantly higher risk of poor outcome (decreased disease-specific survival) compared to patients with tumours expressing little or no PC (Somasiri et al., 2004).PC-positive tumours were not significantly different in histological classification, size or risk of lymph node metastasis.However, PC expressing tumours were more likely to present at an advanced tumour grade, express p53, and be negative for estrogen receptor (ER) (ESR1) and HER-2 (ERBB2) (Somasiri et al., 2004).These data reveal that high expression of PC in primary, locally invasive breast carcinoma is a strong and independent predictor of poor outcome (Somasiri et al., 2004).As with its detection of rare RCC subsets, evaluation of PC expression in primary breast carcinomas may have immediate diagnostic and prognostic value in guiding treatment or surveillance strategies in breast cancer.In addition, detailed profiling of PC-tumours may provide a deeper understanding of tumour progression mechanisms.Related to these efforts, in the next section we discuss some of the molecular mechanisms that underlay PC's role in promoting progression of invasive breast cancer to metastatic breast carcinoma.

Podocalyxin regulates breast cancer tumour cell invasiveness and migration in vitro
In order to gain insight into the biological role of PC in breast cancer, we, as well as other investigators, have surveyed the expression of PODXL and its intracellular ligands, NHERF-1 and -2, in human breast cancer cell lines and examined the effects of altering its expression on the behavior of these cells in vitro and in vivo.Recent microarray analysis of a collection of 50 human breast cancer cell lines revealed a wide range of expression of PODXL, NHERF-1 (SLC9A3R1) and NHERF-2 (SLC9A3R2) transcripts (Kao et al., 2009) (Fig. 1).PODXL tends to be highly expressed in estrogen receptor-and progesterone receptor (PR)-negative (ER - /PR -) basal-like breast cancer lines.Conversely, high NHERF-1 and NHERF-2 expression correlates with luminal-type, ER + breast cancers.MCF7 cells are a line of luminal-like breast epithelial cells (Neve et al., 2006;Kao et al., 2009) obtained by pleural effusion (i.e., lung metastases) of a patient with metastatic invasive ductal adenocarcinoma (ER + /PR + ) (Soule et al., 1973).Relative to other breast cancer cell lines, MCF7 expresses low-moderate levels of endogenous PODXL transcripts and high levels of NHERF-1 (and low to moderate expression of NHERF-2) (Kao et al., 2009) (Fig. 1).MDA.MB.231 cells, which express high levels of endogenous PODXL, are a basal B-subtype breast cancer cell line derived from pleural effusion of human metastatic breast adenocarcinoma (Kao et al., 2009) (Fig. 1).In contrast to MCF7 cells, MDA.MB.231 are ER -/PR -and have a highly invasive phenotype in in vitro assays and in vivo xenograft models (Lacroix and Leclercq, 2004;Neve et al., 2006).By overexpressing exogenous PC and structural mutants in MCF7 cells or by suppressing endogenous PC in MDA.MB.231 cells using RNA-interference (RNAi) methods, we (and others) have found that PC promotes a more aggressive, invasive phenotype in vitro and in vivo (Somasiri et al., 2004;Sizemore et al., 2007).In addition, our preliminary results corroborate a role for PC in breast cancer invasion since suppression of endogenous PODXL in MDA.MB.231 impairs serum-dependent migration in vitro (Turvey & McColl, unpublished observations).We are currently working to determine if silencing PODXL expression down regulates invasion-associated adhesion molecules and matrix proteinases and how loss of PODXL alters the morphology and cytoskeletal dynamics in MDA.MB.231.The transcript expression heat map was generated from meta-analysis of publically available microarray gene expression profiles of human breast cancer cell lines (Kao et al., 2009) using Gene Cluster 3.0 and Java TreeView.

Podocalyxin modulates EMT-independent breast cancer progression and metastatic potential
Epithelial-to-mesenchymal transition (EMT) is an important paradigm in the understanding of tumor progression to systemic, metastatic cancer (discussed in more detail in §3.5.1).However, EMT is not an obligate pathway to metastases (Wicki et al., 2006), and invasive ductal breast carcinomas, which form the great majority of locally invasive breast lesions, retain epithelial characteristics and epithelial differentiation markers (Cleton-Jansen, 2002;Sarrio et al., 2008).Thus, a better understanding of EMT-independent mechanisms of tumour invasion and metastasis is critical for developing therapeutic strategies in ductal breast carcinoma in particular, but also other cancers in general.By expressing exogenous PODXL in MCF7 cells, we have found that PC promotes altered morphogenesis, loss of adhesion and impaired tumour cell aggregation in 3D spheroid cultures (Graves and Roskelley, manuscript in preparation).Importantly, altered adhesion is not associated with disruption of cell-cell junctions, impaired expression or re-localization of cell-cell junction regulators (e.g., E-cadherin, -catenin, ZO-1) or, disruption of extra-cellular matrix (ECM) adhesion complexes (e.g., 1-integrin, activated focal adhesion kinase (FAK)).Rather, expression of PC in polarized cells enhances the exclusion of 1-integrins from the free, apical surface of MCF7 and recruits F-actin from the basolateral ECM-adhesion complexes to an expanded apical membrane domain.We hypothesize that PC-mediated redistribution of Factin and other cortical actin components weakens adhesion of the basolateral membrane domain to ECM.Moreover, when single cells are shed from the epithelial sheets, PC is redistributed in a global pattern that blocks homotypic adhesion.Expression of PC on shed, single cells may also promote chemotaxis, enhance adhesion-free survival or participate in the homing to distant tissue sites (Fig. 2).Thus, while PC expression in some epithelial tissues may help to define the apical domain and reinforce polarization, dysregulated mistargetting or overexpression of PC disrupts normal tissue architecture of epithelial sheets and spheroids and may promote invasive and metastatic characteristics.We are continuing to use these models, and orthotopic mammary fat pad transplant breast tumour models, to fully characterize PC's role in breast tumour invasion and metastases.In summary, the data suggest PC plays a key role in enhancing breast carcinoma growth and invasiveness in vivo.

Ovarian carcinoma
Ovarian cancer remains the most lethal gynecological cancers and, like most epithelial carcinomas, systemic spread generally has a poor prognosis since there are currently no curative interventional therapies.PC is expressed in normal mesothelia-derived tissues that encase the ovaries and make up the free luminal surface of cells that line the oviducts and endometrium (Cipollone and Roskelley, manuscript submitted).In addition, PC protein is detectable in the tumor cells of approximately two-thirds of human ovarian carcinoma cases.Although the mere presence of PC protein in ovarian carcinomas does not correlate with disease outcome, expression of PC on the cell surface (as opposed to intracellular localization) is predictive of poor outcome for high-grade serous ovarian carcinoma, 87% of which express significant amounts of the mucin.As with breast cancer cell lines, PC expression in ovarian cancer lines is variable (Cipollone and Roskelley, ibid).To evaluate PC's molecular functions in ovarian carcinoma, we ectopically expressed PC in the serous carcinoma-derived cell line (OVCAR-3) originally derived from malignant ascites (Hamilton et al., 1983).OVCAR-3 cells express epithelial differentiation markers, form well-structured adherent epithelial sheets in culture, and are generally noninvasive and have low motility in vitro (Comamala et al., 2011).Forced expression of PC in OVCAR-3 cells reduced in vitro adhesion to extracellular matrices including; mesothelial-cell layers, immobilized 1-integrin antibodies and fibronectin.As with other epithelial cells, exogenously expressed PC is preferentially localized to the apical free surface of OVCAR-3 cell layers in 2D culture and spheroids in 3D culture.As observed in MCF7 cells, PC excludes 1integrin from free apical domains.Thus, when expressed at the cell surface, PC may act as an anti-adhesin in serous ovarian carcinoma by sterically masking integrin-extracellular matrix interactions, and, by promoting cortical cytoskeletal rearrangements that weaken adhesion to ECM.This PC-induced morphogenesis may be of critical importance to ovarian carcinoma progression, as these tumors very often metastasize after small nodules are shed, in an antiadhesive (non-EMT dependent) fashion, into the abdominopelvic cavity.Thus, while PCdriven non-EMT shedding and migration may be rare in some epithelial cancers (breast and renal), PC expression may be a common, and critically important molecular mechanism driving tumour metastasis in high-grade serous ovarian carcinoma.

Thyroid carcinoma
Thyroid carcinomas (non-lymphoma, squamous cell or sarcoma) are classified according to histological characteristics and origin.The most common "differentiated" subtypes (papillary and follicular) are highly treatable and responsive to therapy.The medullary and undifferentiated (anaplastic) sub-types, although more rare, have a less favorable prognosis.A l t h o u g h P C i s n o r m a l l y a b s e n t o n t h y r oid epithelia and is not detected in welldifferentiated thyroid carcinoma tumour subtypes or squamous cell carcinoma (thyroid), in immunohistological analyses of 238 thyroid tumours, PC was detected on tumours of over half the cases (n=69) of undifferentiated thyroid carcinoma (UTC).In addition, in cases where the thyroid tumours have a mixed histological type (UTC adjacent to differentiated tumour cells), PC was detected only on cells with UTC phenotype (Yasuoka et al., 2008).We note that the pattern of PC expression on thyroid carcinoma and small-cell lung carcinoma ( §3.5) (correlates with a "de-differentiated" tumour phenotype consistent with an EMT transformation.Thus, although PC may not be sufficient to drive EMT, high expression on UTC and other mechanistic studies ( §3.5.1) suggest that PC expression may be upregulated as part of an EMT program.If this is so, similar to its function in EMT-independent tumours, PC may help to promote a non-cohesive, invasive phenotype during EMT.

Small-cell lung carcinoma
Small-cell lung carcinomas (SCLC) (16% of lung cancers) are malignancies that typically arise from epithelial cells of the proximal airways (bronchus).SCLC tumours consist of multipotent epithelial ("stem-like") cells that form typically diffuse and non-cohesive clusters.Analysis of formalin-fixed, paraffin-embedded bronchial-tumour biopsies (and adjacent normal bronchial tissues) reveals that the majority of SCLC but not normal bronchial epithelia expresses PC and its expression in SCLC correlates with non-methlyated CpG islands in the PODXL gene (Koch et al., 2008).Although PC is not detected in normal adult bronchial epithelium, clusters of PC-positive epithelial cells can be detected in deep pockets of developing proximal bronchi and trachea of fetal lung (Koch et al., 2008).The authors suggest the intriguing hypothesis that PODXL marks multipotent bronchial epithelial cells during lung development and that SCLC "cancer stem cells" may retain or acquire the characteristics of this stem-like population.There are currently no studies that correlate PC expression with patient outcome or response to chemotherapy in lung cancers.PC expression in non-small cell lung carcinomas (NSCLC) was not performed in this study so it is not yet known if PC also marks primary NSCLC tumours of the lung.For these reasons, the diagnostic value of PC expression in lung cancer is less certain than in other cancers.However, although many technical challenges remain, identification of tumour-specific glycoforms of PC (see following sections) may provide targets for adjuvant therapy in lung cancer.This is particularly important in SCLC where surgical resection of tumours is more difficult.Alternatively, if PC expression is consistently associated with type 3 EMT processes in lung cancer (see below), or other EMT-dependent cancer metastases, expression of PC (assessed by biopsy or in vivo imaging) may herald a switch from locally invasive to a more advanced disease stage.

Podocalyxin in TGF-mediated epithelial-to-mesenchymal transition (EMT)
Epithelial-mesenchymal transition (EMT) (type 3) is a paradigm of tumour invasion and metastasis whereby epithelial cells lose their apical and basal polarization and robust cellcell contacts to become mesenchymal in morphology and adopt an invasive, migratory behaviour (Kalluri and Weinberg, 2009).Among many other alterations in gene expression patterns, EMT is associated with loss of the adherens-junction regulator E-cadherin (CDH1), stabilization and nuclear-localization of -catenin (CTNNB1), and upregulation of the intermediate filament protein vimentin (VIM), a mesenchymal marker (Zeisberg and Neilson, 2009).As we have noted above, PC does not appear to be capable of initiating EMT on its own.However, Wilkins et al. have recently demonstrated that it may contribute to the EMT that is initiated by transforming growth factor beta (TGF (Meng et al., 2011).Although TGF has tumour suppressing activity in some cancers, especially at the early stages of tumour progression, TGF, in the context of enhanced phosphatidylinositol-3-kinase (PI3K)/Akt and other survival signalling pathways can drive EMT processes (Ouyang et al., 2010).TGF-induced de-differentiation is not limited to malignant cells, as normal human and mouse podocytes undergo an EMT-like transition in response to TGF (Herman-Edelstein et al., 2011;Li et al., 2008;Wang et al., 2011).A549, a human lung adenocarcinoma cell line, undergoes EMT-like transformation in the presence of TGF, with prototypical EMT features including down regulation of E-cadherin, upregulation of vimentin, expression of MMP2 and fibronectin and, secretion of collagens (type I, III and IV) (Kasai et al., 2005).These alterations in gene expression pattern are accompanied with altered cell morphology, loss of cell-cell contact and enhanced migration (Kasai et al., 2005;Meng et al., 2011).Fig. 3. PODXL expression is upregulated during TGF-induced EMT of a lung adenocarcinoma cell line (A549).Expression profiles where generated by meta-analysis of publically available gene expression data using the Gene Expression Omnibus (GEO) dataset GDS3710 (Sartor et al., 2010).
Meng et al recently demonstrated that PC protein expression is upregulated in response to TGF (Meng et al., 2011) A query of a publically available gene expression dataset of A549 cells undergoing TGF-induced EMT corroborates their findings and shows that the kinetics of PODXL transcript expression are similar to EMT-associated down regulation of E-cadherin (CDH1) transcript (Sartor et al., 2010) (Fig. 3).Intriguingly, using a genesilencing approach (shRNA), Meng et al show that PC is required for at least some EMTrelated processes including mesenchyme-like morphogenesis, cell migration and changes in expression of E-cadherin and vimentin (Meng et al., 2011).PC was also found to codistribute with newly secreted collagen I at the leading edge of migrating TGF-treated A549 cells.In addition, collagen I could be co-precipitated with anti-PC antibodies in TGFtreated A549 cells.Notably, the collagen I-binding integrins (11 and 21) were not identified in collagen I-PC complexes or detected in PC-collagen membrane domains.Thus, one possible role for PC in a collagen-complex could be the exclusion of collagen-or matrixbinding integrins from the leading edge of migrating cells and intercellular destabilization of strong adhesion to ECM -themes consistent with our findings using breast and ovarian cancer cell lines ( §3.2.3 & 3.3).We note that in this example, PC expression is down-stream (or parallel) to TGF-induced EMT.Thus, while high expression of PC in some tumour cells (e.g, breast carcinoma) can promote EMT-independent tumour progression, PC may also support the invasive and migratory functions of tumour cells that have undergone EMT.

Prostate carcinoma
A 1.1 Mb region of human chromosome 7q32-33 flanked by microsatellite markers D7S2452 and D7S684 strongly linked to prostate tumour aggressiveness contains the PODXL locus (Neville et al., 2002).In a sibling-pair study, a variable in-frame, germ-line deletion of one or two Ser-Pro repeats in the PC mucin domain (deletion of residues 23-24 or 23-26) was found to be associated with enhanced risk of developing prostate cancer (increased overall risk (OR)=2-2.5fold) and increased tumour aggressiveness (OR=3-4.4fold) (Casey et al., 2006).In addition, a missense single-nucleotide polymorphism (SNP, G340A) resulting in a Ser substitution at Gly114 (also in the mucin domain) was associated with a 50% increased risk of prostate cancer (Casey et al., 2006).Other SNPs in PODXL were detected but had lesser correlative significance to prostate cancer risk or tumour behaviour.We note that, unlike the other cancers discussed in this review, this is the only example of a germ-line mutation in PODXL associated with increased cancer risk.Although 7q32-33 is a region of high allelic imbalance in prostate cancer, this patient study did not specifically address PC protein or transcript expression levels in prostate tumours.Although we may not expect that deletion of one or two glycosylation sites arising from PODXL polymorphisms would significantly alter PC function, it is possible that such in-frame deletions may alter PC surface stability, apical localization or expression and therefore account for enhanced aggressiveness of prostate tumours.Alternatively, in-frame deletions of mucin domain Ser-Pro may indeed alter the functions of PC's extracellular domain or its association with unknown ligands.Regardless of the associated molecular mechanism, PODXL deletion polymorphisms and the G340A missense SNP may provide an additional genetic screening method to evaluate prostate cancer risk.

Forced expression of podocalyxin enhances the motility of prostate cancer cells
PC3 cells are a human prostatic adenocarcinoma cell line derived from bone metastases (Kaighn et al., 1979).PC3 cells, which are considered to have a relatively high metastatic potential and invasive phenotype in comparison to other prostate cancer lines, do not express detectable levels of PC by western analysis (Sizemore et al., 2007).However, as for other epithelial tumour cell lines, forced expression of full-length PC in this prostate cell line enhanced cell motility in vitro (Sizemore et al., 2007).This enhanced motility may depend on the activation of Rac1 that, in turn, requires the association with two intracellular binding proteins, NHERF1 and ezrin.Thus, as for other carcinomas discussed in this review, expression of PC in prostate epithelial has the potential to impart a more aggressive phenotype with potential clinical importance.Further study of PC expression profiles in primary or secondary prostate tumors, coupled to tumour phenotype and outcome data, are required to evaluate whether expression of PC has diagnostic value in prostate cancer.Query of microarray gene expression data from normal prostate tissue and primary and secondary prostate cancer suggest that PODXL expression may not be altered in primary prostate tumors (Fig. 4).Conversely, metastatic tumours originating from the prostate may have increased PODXL expression (~1.4 fold) (Fig. 4).Therefore, it is unclear if the increased risk and tumour aggressiveness associated with this PODXL polymorphism is related to altered PC function, expression or simply another marker that is linked to allelic imbalance of 7q32-33.(Chandran et al., 2007;Yu et al., 2004).The metastatic tumour group is significantly different than the normal adjacent and primary tumour groups but not the normal donor group (*p < 0.05, Tukey one-way ANOVA analysis).All other group comparisons are not significantly different.

Testicular cancer and embryonal carcinoma
Testicular cancer, the most common cancer in young males, has a high cure rate even after systemic spread (reviewed in Winter & Albers, 2011).However, risk stratification and classification of the primary tumour by histological methods and determination of tumour marker levels is important for designing surveillance and systemic treatment strategy in the event of metastasis or re-lapses.The vast majority of testicular tumours (95%) can be broadly classified as either seminomatous-(SGCT) or non-seminomatous-(NSGCT) germ cell tumours.Seminomas are non-pluripotent, undifferentiated germ cell progenitors whereas nonseminomas tumours are comprised of several subtypes arising from embryonic or extraembryonic (e.g., yolk sac) lineages.Embryonal carcinomas and teratomas are examples of sub-types of NSGCT.Importantly, treatment strategies for SGCT and NSGCT cancers are different, with the major difference being that NSGCTs are generally resistant to radiotherapy.PC is antigenically identical to tumour markers originally designated GCTM-2 (or gp200), TRA-1-60 and TRA-1-80 expressed on embryonal carcinoma (EC) cell lines and inner blastocyst-derived human embryonic stem cells (hESCs) (Schopperle et al., 2003;Schopperle & DeWolf, 2007).The TRA-1-60/81 epitopes are present only on a high molecular weight form of PC (200 kD) expressed by undifferentiated embryonal carcinoma or hESCs.Intriguingly, the antigenic sites detected by these antibodies disappear following retinoic acid (RA)-induced differentiation of ECs or following differentiation of ESCs into embryoid bodies (EBs) (Schopperle and DeWolf, 2007).However, EBs continue to express a lower molecular weight isoform (170 kD) of PC that can be detected using antibodies recognizing peptide epitopes, or by the terminal glycan, -(D)-galactose (Gal)-binding plant lectin, peanut agglutinin (PNA) (Schopperle and DeWolf, 2007).Thus, there is evidence to suggest that there are tumor and tissue type specific epitopes on PC.These epitopes are present on primitive or pluripotent germ-line tumours or hESCs.GCTM-2 has been identified as a tumour marker of NSGCT and a soluble form of GCTM-2 has been detected in the sera in 7 of 20 patients diagnosed with NSGCT.In addition, TRA-1-60 was present in the sera of over 75% of patients (n=42) with NSGCT and even in twothirds of NSGCT patients negative for the "gold-standard" testicular cancer serum tumour markers, -fetoprotein and chorionic gonadotrophin (Mason et al., 1991;Badcock et al., 1999;Gels et al., 1997as cited in Schopperle et al, 2003).Although the mechanism has not yet been defined, serum-soluble forms of PC may arise from secreted isoforms or proteolytic cleavage (membrane shedding) of the membrane anchored protein.These findings suggest that PC; especially a modified, high molecular weight form preferentially expressed in undifferentiated embryonic lineages, may serve as a sensitive tumour marker detectable in patient serum.Thus, with further validation, the detection of soluble PC peptide fragments could aid in detection of testicular germ-line tumours and evaluating response to therapy.

Epigenetic regulation of podocalyxin by micro RNA in testicular cancer
In a screen of gene methylation patterns in the a human testicular tumour cell line (NT2) (an embryonic carcinoma), Cheung et al (Cheung et al., 2011) recently identified a hypermethylated region in intron 14 of the dynamin 3 locus (DNM3) located on chromosome 1(q24).This region codes for the expression of three micro RNAs (miR), including miR-199a2 (hsa-mir-199a-2) in an antisense orientation.miR-199a2, together with miR-199a-1 (located on hChr 19p13), encode the pre-microRNA species miR-199a.Reduced expression of miR-199a has previously been linked to poor prognosis in serous ovarian carcinoma (Nam et al., 2008) and pro-inflammatory and anti-apoptotic pathways in chemoresistant ovarian cancer stem cells (Yin et al., 2010).miR-199a, which is normally abundantly expressed in most human epithelial and non-epithelial tissues (with the notable exception of brain and peripheral blood mononuclear cells (Liang et al., 2007)), targets a cluster of genes that include transcripts encoding PC, HIF-1, IKK and SIRT1, mTOR, SMAD1 and c-MET and several others (Cheung et al., 2011).Note that regulation of these targets by miR-199a and expression of the miR-199a-1 and miR-199a-2 loci is tissue specific.For example, reduced expression of miR-199a (along with other miRs) has also been linked to bladder cancer (Ichimi et al., 2009).Conversely, miR-199a expression is upregulated with a cluster of other miRs in metastatic uveal melanoma and gastric cancer (Ueda et al., 2010;Worley et al., 2008).The miR-199a2 pre-microRNA is processed to yield two mature miR species, miR-199a-5p and miR-199a-3p, and both are down regulated in malignant testicular tumours in comparison to normal or benign tumour tissue (Cheung et al., 2011).miR-199a-5p and -3p have distinct RNA targets, and only miR-199a-5p is a direct negative regulator of PODXL.Expression of miR-199a-5p negatively correlates with PC protein expression in malignant testicular tumors (both seminomatous and non-seminomatous) (Cheung et al., 2011).Ectopic expression of miR199-a in NT2 cells attenuates migration and invasion in vitro and tumour growth and metastatic potential in xenograft transplant models.Suppression of PODXL in NT2 cells by RNAi also attenuates matrix invasion in an in vitro assay.These data suggest that epigenetic regulation of the ha-miR-199a-2 locus by CpG methylation results in repression of miR-199a2 expression and upregulation of PODXL and that these events correlate with tumour malignancy in testicular cancer.Other targets of miR-199a-5p and -3p are also likely involved in the invasive phenotype of testicular tumours.

Gastrointestinal cancers 3.8.1 Hepatocellular carcinoma
Hepatocellular carcinoma (HCC) is a common adult liver malignancy associated with infection by hepatitis (HVB and HVC) and cirrhotic liver disease.Patients with cirrhosis from a variety of causes are at an increased risk of developing HCC.Unfortunately, the majority of HCC patients (>80%) present with systemic or locally advanced tumours that cannot be cured surgically.Although recently, the application of anti-angiogenic drugs (e.g., sorafenib) has shown promise in slowing tumour growth in HCC patients, chemotherapy and radiotherapy are ineffective, and HCC has a very low five-year survival rate (~5%).Prompt detection and diagnosis of HCC in cirrhotic liver by imaging methods or histological assessment of biopsy is critical for designing an effective treatment strategy.Although one group showed that PC was only weakly present in 9% of HCC tumours (i.e., neoplastic liver epithelial cells) (Ney et al., 2007), PC and CD34 were commonly upregulated on sinusoidal endothelia associated with primary HCC tumour tissue (Chen et al., 2004;Heukamp et al., 2006).In contrast, in normal liver tissue, PC is restricted to hepatic arterioles (Heukamp et al., 2006).PC and CD34 are also present (in a more punctuate pattern) on the sinusoidal endothelia of hyperplastic focal nodules and liver adenomas (Heukamp et al., 2006).Thus, expression of PC (and CD34) may provide an additional diagnostic marker for the evaluation of liver biopsies in HCC.PC and CD34 are widely expressed in vascular endothelia in adult tissues but their function remains ill-defined.Although PC has been shown to play a role in the formation of vascular lumens in the developing embryo, it is not essential for the homeostatic maintenance of vascular endothelia in adults (at least in mice).Nevertheless, mounting evidence suggests that PC and CD34 have a role in the formation of new vessels during development and vascularization of solid tumours; and, the maintenance of vessel integrity in inflamed tissues (Blanchet et al., 2008;Maltby et al., 2011;Strilic et al., 2009).Thus, further study of the role of CD34 and PC in neovascularization and vessel homeostasis may provide new targets for anti-angiogenic treatments for solid tumours.

Pancreatic ductal adenocarcinoma
Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic cancer, often detected at an advanced stage.Because there are no effective systemic therapies, PDAC diagnosis typically has a very poor prognosis (~3% 5-yr survival).Determining the primary source of adenocarcinoma is important in treatment strategies and prognosis.Neoplasms originating from the pancreatic duct, gall bladder, bile duct, ampulla of Vater and duodenum are difficult to distinguish by immunohistology alone since many express a similar profile of epithelial cytokeratin-and mucin-family proteins.Immunohistochemical analysis of primary adenocarcinoma and carcinoma tumours of gastrointestinal, pancreatic and extrapancreatic origin suggest expression of PC is associated with primary epithelial neoplasms of the pancreatic duct (44%) or ampullary origin (30%) (Ney et al., 2007).Although PC is expressed is less than half of these adenocarcinomas, it is not detected (or very weak) in normal or inflamed pancreas (chronic pancreatitis) or in most primary tumours from all other gastrointestinal sites including bile duct.Although these data suggest that expression of PC may be useful for the differential identification of primary adenocarcinoma, it is not known if PC expression at secondary tumour sites correlates with their primary origin.However, expression of P C d o e s a p p e a r t o c o r r e l a t e w i t h m o r e advanced histological tumour grade, as 53% of Grade 3, 48% of Grade 2 and 18% of Grade 1 tumours express PC (Ney et al., 2007).Thus, as with several other epithelial malignancies described in this review, expression of PC correlates with a more anaplastic, poorly differentiated phenotype and more aggressive (or advanced) cancers.

Colorectal cancer
Although the expression of PC has not been verified in primary or metastatic tumours in human colorectal cancer, two studies have identified PC expression in colon cancer cell lines, and PC protein as a shed, soluble component of colon cancer cell cultures (Ito et al., 2007;Thomas et al., 2009) In addition, Thomas et al (Thomas et al., 2009) demonstrated that PC, immunopurified from a colon cancer cell line (LS174T), can act as an E-and L-selectin ligand (but not P-selectin) using a PC-coated microbead flow adhesion assay.E/L-selectin binding was dependent on a sialofucosylated epitope of PC expressed in LS174T, similar to, but distinct from sulfated sLe x epitopes present on sialomucins processed in HEV (see §5.2) (Thomas et al., 2009).Thus, tumor-specific modifications (glycosylation) of PC may allow for interaction with selectins.Importantly, neither of these studies showed that PC protein is expressed on the surface of colon carcinoma cells (as opposed to intracellular), or that PC serves as an E/L-selectin adhesive ligand in vitro or in vivo.If PC were indeed shown to be a bona fide E/L-selectin ligand on these carcinomas, this would offer an attractive target for the development of adjuvant therapies.This is especially true if the unique modification of PC detected in human colon cancer cell lines is recapitulated in primary and metastatic colorectal adenomas and carcinomas.Retrospective analyses of tissue microarrays of primary colorectal carcinomas and adenocarcinomas (and perhaps metastatic, secondary tumour sites) similar to those performed for RCC and breast cancer are required to determine if expression of PC is associated with colorectal cancers and if expression correlates with patient outcome.At least one study (Ney et al., 2007) suggested that PC is not commonly expressed in gastrointestinal tumours, including colon-derived, with the exception of pancreatic cancers.Then again, this is also true for breast and renal cancer -the prognostic power of evaluating PC expression in human cancer appears to be in identifying rare, highly lethal sub-types.

AML, ALL and cutaneous myeloid sarcoma
PC expression has been reported on a human monocytic cell line (U937) after induced differentiation with vitamin D3 and transforming growth-factor beta (TGF) and on normal human monocytes treated with macrophage colony-stimulating factor (MCSF) for 3-6 days in vitro (Riccioni et al., 2006).In both cases, PC expression correlates with a more differentiated, monocytic cell-surface marker phenotype (e.g., CD14 + CD11b + ).However, PC expression has not been reported on human monocytes derived from normal bone morrow (BM) or peripheral blood (Kelley et al., 2005).Kelley et al (Kelley et al., 2005) found that PC is often expressed on leukemic blasts including 77% (n = 39) of acute myeloid leukemia (AML), 81% (n = 27) acute lymphoid leukemia (ALL) and 87% (n=15) cases of cutaneous myeloid sarcoma (Kelley et al., 2005).Expression of PC was detected in all clinical sub-types of AML (M1-M7) and both T and Bcell lineage ALL.A second study reported a much lower frequency of PC-positive blasts in 73 cases of AML with approximately 20% of cases presenting with a moderate frequency of PC-positive cells (20-50% positive blasts (+)) and 18% displaying high frequency (>50% positive blasts (++)) (Riccioni et al., 2006).Importantly, while Kelley et al used immunohistochemical analysis of formalin-fixed BM biopsy tissue and PC was observed in a punctuate, cytoplasmic distribution; Ricconini et al used freshly isolated BM or peripheral blood-derived leukemic blasts and flow cytometery analysis (i.e., cell-surface expression of PC).Thus, while PC is commonly expressed in leukemic blasts in the BM, cell-surface exposure of PC is likely more rare (at least in AML).
There are no studies that address the clinical consequences of PC expression in leukemia.Expression of PC in hematopoietic malignancies may reflect transformation of an immature progenitor that normally expresses PC or a "de-differentiation" gene expression pattern of a leukemic stem cell.We have found that expression of PC in mouse hematopoietic stem or progenitor cells (HSC/Ps) enhances CXCR4-induced chemotaxis and BM-homing/retention (see §6.2.3) (Nielsen & McNagny, 2009a) (and our unpublished observations).Thus, one possible consequence of PC overexpression in leukemia may be that PC enhances close contact to CXCL12-secreting cells and, subsequently, an advantage (over normal hematopoietic progenitors or PC-negative leukemia) with to respect retention, proliferation, survival or chemo-resistance of leukemic cells.

Malignant astrocytic tumours
Astrocytomas, tumours originating from astrocytes of the brain and spinal cord, are the most common neoplasm of the central nervous system (Zhu & Parada, 2002).Often presenting at an advanced stage, astrocytic tumours are classified into four histological grades that broadly include benign (I), diffuse (low-grade) (II), anaplastic astrocytoma (III) and glioblastoma (IV).The most aggressive, glioblastoma (GBM), is also the most common malignant astrocytic tumour with a 5-year survival rate of less than 3%.Although systematic metastasis of astrocytomas is rare, aggressive local invasion into surrounding tissue, rapid growth and resistance to apoptosis accounts for the lethality of these brain cancers -tumour behaviours supported by aggressive angiogenesis (Anderson et al., 2008).In a study of 51 astrocytomas using immunohistochemical staining of frozen sections, immunoblotting of tumour lysates and quantitative real-time PCR, PC expression was found to be present in approximately half of the cases of anaplastic astrocytoma and GBM, but none of the lower-grade astrocytomas (Hayatsu et al., 2008).The pattern of staining in the anaplastic astrocytomas is consistent with cell-surface expression of PC; however, PC localization in GBM was more diffuse and PC-positive tumour cells were associated with regions of microvascular proliferation.Using a lectin-binding microarray combined with a mass spectrometry approach, another group detected PNA-reactive PC abundantly expressed on the surface of a "stem cell like" human GBM cell line (GBM1), but not on an adherent GMB line (U373) (He et al., 2010).Notably, the sialomucin podoplanin (PDPN), which shares some striking structural and functional properties with PC, is also highly expressed in glioblastoma (Mishima et al., 2006).Podoplanin is expressed on the outer/invasive edge of some tumours and is thought to have a role in the anti-adhesive phenotype of EMT-independent tumour invasion (Wicki and Christofori, 2007).Invoking a similar paradigm to breast and ovarian carcinoma ( §3.2.3 & 3.3), we posit that high expression of PC may promote an EMT-independent morphogenesis that, combined with other transforming pathways, promotes an invasive phenotype in astrocytomas.

Podocalyxin marks embryonal carcinomas and embryonic stem cells
PODXL transcript and protein is down regulated in embryoid bodies (EB) and lineagerestricted EB cultures compared to undifferentiated human embryonic stem cells (hESCs) (Brandenberger et al., 2004;Cai et al., 2006).PC-binding antibodies TRA-1-60 and TRA-1-81, together with membrane surface, stage-specific embryonic antigens (SSEA)-3 and SSEA-4; and, intracellular markers alkaline phosphatase, telomerase, Oct4 and Nanog are routinely used to characterize undifferentiated hESC (De Miguel et al., 2010;Palecek, 2011).As mentioned previously, the TRA-1-60 and TRA-1-81 epitopes are present only on the high molecular weight form of PC detected in embryonal carcinoma (EC) cell lines and these markers are lost upon induced differentiation of EC or hESC.Thus, PC is a marker of undifferentiated human embryonal cells and PC is either alternately modified (e.g., glycosylation pattern) or its expression is reduced upon differentiation.In an effort to identify novel markers of undifferentiated hESC, Choo et al generated an anti-PC antibody (IgM isotype, mAb 84) cytotoxic to undifferentiated hESC (human embryonic stem (HES) cell lines HES-2, -3 and -4) and at least one embryonal carcinoma cell line (NCCIT) (Choo et al., 2008).Importantly, mAb 84 is not cytotoxic to mature human cell lines HeLa) or hESC induced to differentiate by withdrawal of fibroblast growth factor 2 or following culture of hESC to form EB (day 22).Remarkably, mAb 84 rapidly (< 45 minutes) kills EC even at 4°C.The mechanism for mAb 84 cytotoxicity is not dependent on complement activation, hypercrosslinking of bound antibody or antibody internalization.Instead, mAb 84 mediates cell death by oncosis initiated by PC-mediated reassembly of cytoskeleton, degradation of cytoskeletal structural proteins (-actinin, talin and paxillin), PC-aggregation and formation of large membrane pores (Tan et al., 2009).Since crosslinking other PCspecific antibodies bound to hESCs is not cytotoxic, the association of mAb 84 to a unique PC epitope (or potentially other additional unknown epitopes on hESCs) induces cytoskeletal reassembly in advance of PC aggregation and pore formation.Studies with antibody fragments revealed that cytotoxicity requires divalency with sufficient flexibility linking the antigen binding domains (Lim et al., 2011).Since pre-treatment of HES-3 hESC and NCCIT cultures with mAb 84 in vitro prevents teratoma formation in a severe combined immunodeficient (SCID) mouse xenograft model mAb 84 has potential applications in clearing tumour-forming undifferentiated hESCs from cultures of differentiated hESCs intended for cell-based, regenerative therapies (Choo et al., 2008;Tan et al., 2009).With respect to the treatment of cancer, a cytotoxic antibody like mAb 84 may also prove to be an effective oncolytic adjuvant therapy in germ-line tumours like testicular cancer (especially embryonal carcinoma subtype).This approach, of course, would require highly selective targeting of tumour-specific forms of PC to avoid renal toxicity.The encouraging example of mAb 84 suggests that highly selective targeting of PC expressing tumour cells in vivo may be possible.

Podocalyxin structure and function
In an effort to determine how expression of PC on cancer cells promotes tumour aggressiveness and metastasis, several groups, including our own, have performed structure-functional analyses in model cancer cell lines to delineate the specific roles of PC's protein domains in the regulation of cell morphology, motility and survival.The following sections summarize some of what is known of the molecular mechanisms regulated by PC.PC's highly charged and bulky extracellular domain imparts a biophysical anti-adhesive property that regulates cell morphology and cell-cell adhesion functions and, when appropriately modified, may also promote adhesion by binding to L-or E-selectin.The intracellular domain links apically-polarized PC to the cortical actin cytoskeleton.By recruiting ezrin and NHERF-1/2, PC's intracellular domain localizes signalling complexes at apical membrane domains.In general, expression of PC promotes cell motility and invasion functions in epithelial cells and promotes chemotaxis in hematopoietic cells.

Podocalyxin is a CD34-family sialomucin
Podocalyxin is one of three members of the CD34-family of sialomucins that also includes CD34 and endoglycan (reviewed in Furness & McNagny, 2006).Mucins are members of a large class of secreted and type I transmembrane proteins that contain at least one extracellular domain rich in proline, serine and threonine residues (PTS domain).Mucin domains are extensively modified by post-translational addition of O-linked oligosaccharides (at S/T residues) and frequently modified by N-linked glycosylation and sulfation.Sialomucins, in particular, are also heavily modified by the addition of a terminal sialic acid to terminal O-linked glycans.PC is a sialomucin that, together with CD34 and endoglycan, makes up the CD34-family (reviewed in Furness and McNagny, 2006).The mature, cell surface expressed human PC protein is a 536 residue glycoprotein with a calculated peptide mass of 56 kilodaltons (kD); however, glycosylation and other post-translational modifications yield a product with an apparent molecular weight of 150-200 kD.The extracellular domain of PC consists of a heavily O-linked glycosylated, sialated and sulfated mucin domain, a globular domain containing a conserved fourcysteine (Cys) motif, and, a membrane-proximal stalk domain.Three or four sites of Nlinked glycosylation also decorate the extracellular domain.Together, these modifications result in a highly negatively charged, sterically bulky glycoprotein (Fig. 5).Although the primary amino acid sequence of PC's extracellular domain is not well conserved by paralogs (or CD34-family homologs), the intracellular domain is highly conserved across species.

Extracellular ligands of podocalyxin
The only well-established extracellular ligand for CD34-family sialomucins is L-selectin (reviewed in (Furness & McNagny, 2006;Nielsen & McNagny, 2008)), however there are also reports to suggest that, when appropriately modified, they may also bind E-selectin (Thomas et al., 2009).Selectins are a family of mammalian C-type lectin carbohydratebinding proteins normally expressed by hematopoietic cells (L-and P-) or endothelia (Pand E-) (Laubli & Borsig, 2010).L-selectin is responsible for naïve homing of leukocytes to secondary lymphoid organs; whereas, P-and E-selectins play a predominant role in mediating homing of white blood cells to inflamed (or specialized) tissues.Recognition of CD34-family sialomucins by selectins requires an unusual carbohydrate post-translational modification (sulfated sialyl Lewis-X (sLe X )).Although this modification is present on CD34-family sialomucins expressed by high endothelial venules (HEV) of lymph nodes, it is not typically found on CD34-type proteins expressed by non-lymphoid endothelia (the vast majority of all endothelia).Thus, although selectin-binding functions for CD34 sialomucins (including PC) have been demonstrated in vitro, the biological significance of this function is unclear for most endothelium in vivo.Nevertheless, the interaction of tumour cells with selectins (or expression of selectins or selectin ligands by tumour cells) is an important paradigm in cancer metastases (reviewed in Laubli & Borsig, 2010).Thus, aberrant modification (sLe X ) or enhanced expression of PC in malignant cells (Kannagi, 2004) may facilitate selectin-mediated tissue homing and trafficking.

NHERF-1 and NHERF-2
NHERF-1 and -2 both have two tandem PDZ domains -the centrally located PDZ class II domain associates with PC's C-terminal DTHL motif, leaving NHERF's N-terminal PDZ class I domain free to interact with other binding partners.Table 2 provides a partial list of known NHERF-1/2 binding partners.Ezrin can either directly binds to PC via the membrane proximal ERM protein-binding motif or indirectly via the C-terminal ERM motif of NHERF-1/2.NHERF-1/2 have a wide range of binding partners that include ion channels, G protein coupled receptors, receptor tyrosine kinases (RTKs), intracellular signalling intermediates and -catenin, a transcription factor (Table 2).NHERF-1/2 may also homodimerize or polymerize via PDZ-domain interactions, or bind other PDZ domain containing proteins like PDZ K1 (Garbett et al., 2010).

Ezrin
Ezrin acts as an intracellular adaptor to provide linkage of integral membrane proteins with the cytoskeleton.Ezrin contains an N-terminal FERM protein-interaction domain capable of binding the intracellular domain of several type I transmembrane proteins, NHERF-1/2, RhoGDI and the membrane lipid, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ), and a C-terminal domain that bind polymerized actin (F-actin) (Table 2).The C-terminal domain of ERM folds over to associate with its N-terminus in an inactive conformation and phosphorylation of a conserved Thr residue in the C-terminus, downstream of Rho kinase, protein kinase C isoforms (and other S/T kinases), induces an open conformation that permits ERM proteins to link complexes to polymerized actin (F-actin) (Fehon et al., 2010).Phosphorylation of Tyr residues (perhaps by RTKs or Src-family kinases) further enhances ezrin activation or activation of ezrin-dependent signalling events.By using NHERFs as an adaptor, ezrin can link several other transmembrane proteins to the cytoskeleton including ion channels and GPCRs.We note that PC is one of few transmembrane proteins that can bind ezrin both directly and indirectly through NHERF-1/2.

The molecular basis for podocalyxin function in tumour metastasis
At least some of PC's molecular functions in normal and neoplastic tissues can be explained by viewing PC as a key electrostatic anti-adhesive force between extracellular membrane surfaces (Gelberg et al., 1996;Takeda et al., 2000;Galeano et al., 2007;Strilic et al., 2009).However, since the intracellular domain of PC is phosphorylated on Ser/Thr and Tyr residues and PC assembles signalling complexes in response to cell activation, PC undoubtedly influences the behaviour of tumour cells by regulating intracellular signalling pathways.Some of these intracellular signalling complexes likely reinforce the localization of PC to the apical membrane of polarized cells.However, they may also enhance (or otherwise regulate) downstream signalling events important in the rearrangement of cytoskeleton and adhesion-independent survival.We propose the extracellular domain of PC is necessary and sufficient for PC's homotypic anti-adhesive functions and morphogenesis of apical membranes.However, stable apical localization of PC, and its effects on the migration and, potentially, survival-promoting functions depends on the intracellular domain and assembled signalling complexes.In the next section we will review the proposed molecular signalling mechanisms regulated by PC and relate these signalling functions to PC's role as a promoter of tumour progression and metastases.

Podocalyxin regulates adhesion and membrane morphogenesis in epithelial cells
Many of the molecular functions of PC, including signalling mechanisms mediated via ezrin and NHERF-1/2, have been elucidated using the human breast cancer cell line MCF7, the human prostate cancer line PC3 and the canine renal tubule cell line, Madin-Darby canine kidney (MDCK).Since human RCC arise from renal tubule cells, the MDCK model cell line may offer valuable insights into the molecular mechanisms of tumour progression and metastasis in RCC.Although renal tubules in human and rabbit do not express detectable levels of PC protein, MDCK cells and adult canine kidney tubular cells have detectable PC (Cheng et al., 2005;Meder et al., 2005).As mentioned in previous sections, MCF7 and PC3 cells are derived from metastatic breast and prostate tumours, respectively.Both of these cell lines express low to moderate levels of PC and are therefore useful for in vitro and in vivo assays using methods to force expression of PC or structural mutants of PC.Conversely, RNAi methods can be used to study the functions of endogenous PC in tumour cells or cell lines.Although there are discrepancies in the assignment of functional roles to PC's structural domains (see details below), taken together, these studies show that high expression of PC results in enhanced motility, invasiveness and destabilization of epithelial cell morphology in 2D-and 3D-cultures.These phenotypes are characteristics of aggressive epithelial tumours with propensity to metastasize.

Podocalyxin's function in epithelial architecture and morphogenesis
Although MCF7 cells express moderate levels of PODXL transcript (Kao et al., 2009) (Fig. 1); by western blotting and immunofluorescence confocal microscopy, expression of PC is low or not detected, respectively (Nielsen et al., 2007;Sizemore et al., 2007;Somasiri et al., 2004).In order to study the molecular function of PC in breast epithelial cells, others and we have ectopically expressed full-length PC and a series of structural mutants in MCF7 (Fig. 6).
Overexpression of full-length PC (FL) in MCF7 results in apical bulging of monolayers, reduced transepithelial resistance (i.e., disrupted cell junctions), delamination and shedding of cells from monolayers in vitro (Nielsen et al., 2007;Somasiri et al., 2004).Strikingly, expression of PC in MCF7 (and MDCK) potently induces formation of apical microvilli.Epithelial microvilli are common features in specialized epithelial layers involved in the secretion or absorption of nutrients, vesicles and proteins from the extracellular space (Lange, 2011).As we anticipated, full-length, exogenously expressed PC recruits NHERF-1, ezrin and Factin to apical membrane domains and forms microvilli in MCF7 cells.However, surprisingly, the intracellular domain of PC is dispensable for the formation of microvilli since the transmembrane-anchored extracellular domain alone (CT) is sufficient to drive morphogenesis and the recruitment of ezrin and F-actin to apical membranes and microvilli in MCF7 (Nielsen et al., 2007).Notably, NHERF-1 is not recruited to apical domains or microvilli in the absence of PC's C-terminal DTHL domain (DTHL or CT).This result suggests that the extracellular domain, likely by a biophysical charge-and steric-based intermolecular repulsion mechanism, can drive membrane morphogenesis and rearrangement of the cytoskeleton (including nucleation of ezrin and F-actin in microvilli) to support stable microvilli.We note that this biophysical mechanism requires high expression of PC since endogenous levels of PC in MDCK and MCF7 are not sufficient to drive microvillus formation.However, such high expression of PC may have relevance in some human epithelial cancers, including the rare sub-sets of high PC-positive metastatic breast cancer and renal cell carcinomas mentioned previously.Although many investigators agree that PC, NHERF-1/2 and ezrin form a complex that marks apical membrane domains, the mechanism of PC's apical targeting is not yet certain.
We have found that expression of full-length PC (FL), and PC mutants CT, DTHL and EC (Fig. 6) in MCF7 result in sorting of these exogenously expressed proteins to the apical membrane domain.Conversely, using a similar series of PC mutants expressed in MDCK cells, two other laboratories show that precise apical targeting of PC requires its intracellular domain (Cheng et al., 2005;Meder et al., 2005).In these studies, although the majority of intracellular-deletion PC mutants were still sorted to the apical domain, only full-length PC with an intact O-linked glycosylation extracellular domain plus an intracellular domain was targeted to apical domains in a way that precisely co-localized with endogenous PC.A GPIlinked PC extracellular domain mutant (GPI-EC, Fig. 6) displayed the most non-restricted membrane localization, suggesting that the transmembrane region of PC may play a role in excluding PC from basal lateral membranes and cell-cell contacts.However, Meder et al show that a non-membrane anchored construct of PC is predominantly secreted from the apical face, thus suggesting that the extracellular domain dictates initial apical sorting of PC in epithelial cells (Meder et al., 2005).Thus, although the extracellular domain of PC may be sufficient for initial apical distribution, the ezrin and NHERF-1/2 binding determinants in the intracellular domain reinforce and stabilize the apical targeting of PC in polarized epithelial sheets.
The above studies were performed by expressing PC constructs in epithelial cell lines that express low to moderate levels of endogenous PC.Using a siRNA-mediated gene-silencing approach to knockdown PODXL in MDCK cells, Meder et al show that endogenous PC is required for appropriate polarization of epithelial sheets and lumen formation in MDCK clusters in a 3D semi-solid culture medium containing extracellular matrix components (Meder et al., 2005).While the parental MDCK line formed organized spheres with a central lumen when suspended in matrix, deletion of PC disrupts this architecture, and MDCK fail to form an organized lumen (Meder et al., 2005).Correspondingly, using siRNA-mediated knockdown of endogenous P C i n M D C K , C h e n g e t a l s h o w t h a t endogenous PC is required for hepatocyte growth factor (HGF)-induced tubulogenesis (Chen et al., 2005).Transfection with siRNA-resistant PC (FL) and mutant constructs reveals that tubule formation of MDCK requires the intracellular domain.However, PC's extracellular O-linked glycosylated mucin domain has a role in refining the architecture of tubule lumens.Finally, enforced expression of PC in MDCK cells inhibits cell-cell adhesion and disrupts junctional complexes.This effect is dependent on sialylation of PC, suggesting a charge-repulsion mechanism.

NHERF-herding function of podocalyxin's intracellular domain
Although the mechanisms responsible for the apical localization of PC require further investigation, it is clear that one of PC's functional roles is the recruitment of adaptor proteins to apical domains.By binding ezrin and NHERF-1/2, PC has the potential to impact the activity or compartmentalization of a variety of signalling pathways.While some of PC's functions can be explained purely by invoking a biophysical charge/steric-repulsion paradigm, several biological functions of PC undoubtedly require recruitment of ezrin and NHERF into apical-domain signalling complexes.Since PC can recruit and restrict NHERF and ezrin (and potentially their numerous binding partners (Table 2)) to apical membrane domains, we posit that many of PC's functions stem from the concentration of signalling complexes -either down regulating signals in other membrane domains (e.g., adherens junctions or focal adhesions) or enhancing signals in the apical domains.Cortactin (CTTN) is an F-actin-and Arp2/3-binding adaptor involved in the branching and nucleation of the cortical actin cytoskeleton and formation of membrane protrusions (reviewed in Weaver, 2008).Cortactin's localization and functional activation is regulated by a large number of binding partners.Importantly, cortactin is enriched in cell protrusions called "invadopodia" and has a role in the invasion of tumour cells through extracellular matrix (Weaver, 2008).PC was found to co-localize with cortactin (and perhaps directly or indirectly bind) at the apical domain of rat podocytes (Kobayashi et al., 2009).Effacement of podocytes caused phosphorylation of cortactin, dissociation of cortactin from PC/apical domains and translocation of cortactin to the basolateral face (Kobayashi et al., 2009).
Although the interaction of cortactin and PC in invasive tumours has not yet been examined, PC localizes to "invadopodia"-like protrusions in in vitro assays.Furthermore, cortactin is known to regulate the secretion of MMPs at invadopodia.In this way, and by recruitment of NHERFs and ezrin, PC may regulate changes in cortical actin assembly associated with tumour invasion and metastasis.This idea is supported by an experiment showing that forced expression of PC in MCF7 enhances expression and secretion of matrix metalloproteinases (MMP)-1 and MMP-9 (Sizemore et al., 2007).
6.2 Podocalyxin's role in the regulation of tumour growth, adherence-independent survival and cell motility 6.

Regulation of Rho-family GTPases
In MDCK cells, formation of a PC-NHERF-1-ezrin ternary complex regulates RhoA activation (Schmieder et al., 2004).As part of a regulatory cycle controlling activation state of RhoA, RhoGDI maintains RhoA in an inactive GDP-bound state.Redistribution of RhoGDI to the apical membrane domain complex PC-NHERF-1-ezrin frees RhoA for activation by guanine exchange factors.Although sequestration of RhoGDI is not dependent on the interaction of PC and NHERF-1 (but does depend on ezrin), activation of RhoA requires NHERF-1 association with PC's C-terminal DTHL motif (Fig. 7).
A recent study corroborates and extends this finding by showing that the PC-NHERF-ezrin ternary complex also regulates activation of Rac1 (a Rho-family member) by a mechanism whereby the N-terminal PDZ1 domain of NHERF-1 recruits the Rho guanine nucleotide exchange factor 7 (ARHGEF7) to this complex via ARHGEF7's C-terminal ENTL (a PDZbinding motif) (Fig. 7) (Hsu et al., 2010).Cell motility requires a coordinated reorganization of the cytoskeleton, a process regulated in part by Rho-family GTPases.By regulating the activity and apical localization of RhoGDI, and ARHGEF7, PC may promote motility in some circumstances.Thus, in addition to dramatic morphogenic changes in the apical domain of epithelial cells resulting in reduced cell-cell and cell-matrix adhesion, inappropriate or unregulated expression of PC may promote cell motility by regulating the activity of small GTPases.Although the above studies were conducted in MDCK cells, others have shown that Rac1 mediates PC-enhanced invasion and motility of PC3 and MCF7 cells (Sizemore et al., 2007).Fig. 7. Podocalyxin enhances activation of Rho-family GTPases by sequestration of RhoGDI and recruitment of a guanine exchange factor (ARHGEF7) to a PC-ezrin-NHERF complex assembled at membrane domains.

ERK and PI3K pathways
Forced expression of PC in MCF7 and PC3 cells induces ezrin-dependent activation of the Akt and Erk1/2 pathways (Sizemore et al., 2007).Moreover, activation of PI3K/Akt and Erk1/2 is required for PC-enhanced invasiveness of MCF7 and PC3.Invasiveness of PCexpressing cells was mediated in part by expression and secretion of matrix metalloproteinase -9 (MMP-9) and MMP-1, which in turn, required expression of ezrin and activation of PI3K and Erk1/2 (Sizemore et al., 2007).Tyrosine phosphorylation of ezrin at central residue (Y353) has been shown to bind the regulatory subunit (p85) of class I PI3Ks and this association, in turn, induces activation of the PI3K pathway (Fievet et al., 2004;Gautreau et al., 1999).In addition, tyrosine phosphorylation of a residue (Y567) in ezrin's C-terminal domain disrupts the active conformation of ezrin and promotes intermolecular interactions with F-actin and other binding partners (Brambilla & Fais, 2009).Expression of PC in MCF7 and PC3 enhances phosphorylation of both the PI3K-activating Y353 and Y567 residues of ezrin (perhaps by Src-family kinases) (Sizemore et al., 2007).Activation of Erk1/2 via ezrin may be more indirect, proceeding through activation of Rho-family GTPases (Sahai and Marshall, 2002).

Podocalyxin enhances CXCL12-mediated migration of hematopoietic cells
CXCL12 (formerly known as stromal derived factor 1 (SDF1)) is a critical chemotatic factor for the maintenance of hematopoietic cells in the BM niche and also for the maintenance of secondary lymphoid organ architecture.CXCL12 is a ligand for two GPCR chemokine receptors, CXCR4 and CXCR7 (reviewed in Teicher and Fricker, 2010).CXCR4 has a wellestablished role in mediating CXCL12 chemotatic cues in cells, whereas CXCR7 does not appear to be a competent signalling receptor on its own, but may regulate CXCR4 activity.Importantly, CXCR4 and CXCR7 expression or function is dysregulated in many human cancers, including (but not limited to) myleloid and lymphoid leukemias and many of the carcinomas discussed in this review.Expression or dysregulated signalling of CXCR4 and CXCR7 promote the metastasis and survival of cancers to (or within) tissues rich in CXCL12 (Sun et al., 2010).
In our efforts to understand the function of PC expression on hematopoietic stem and progenitor cells (HSC/Ps), we have used an RNAi approach to knockdown Podxl expression in a mouse myeloid-progenitor, factor dependent cell line, FDCP-1.We have previously shown that PC is highly expressed in FDCP-1 and, upon stimulation with interleukin-3 (IL3), PC is redistributed from a global membrane expression pattern to a polarized membrane domain that also recruits NHERF-1 (Tan et al., 2006).Recently, we have found that silencing Podxl in FDCP-1 inhibits CXCL12-mediated migration (manuscript submitted).In corroboration of this finding, primary hematopoietic cells derived from Podxl - /-fetal liver (day 15.5 embryos) also display impaired migration to CXCL12 in an in vitro assay.Although we have not yet uncovered the molecular mechanisms underlying PCenhanced migration to CXCL12, we have found that stimulation of FDCP-1 (or mouse fetal liver) with stem cell factor induces enhanced surface exposure CXCR4 that then becomes distributed with PC to a common polarized membrane domain.This distribution suggests that PC and CXCR4 can physically associate at the cell surface.Indeed, CXCR4 can be coprecipitated with PC antibodies under some conditions (unpublished observations).We hypothesize that PC, as exemplified by the related sialomucins CD34, PSGL-1 and CD164 (endolyn) (Forde et al., 2007;Veerman et al., 2007;Blanchet et al, 2011), has an active role in hematopoietic cell migration and tissue homing by stabilizing or enhancing chemokine receptor signalling.Although we currently favour a mechanism by which PC associates with, and physically stabilizes, CXCR4 at a polarized membrane domain, PC may also enhance intracellular CXCR4 signalling pathways including activation of Rho GTPase, PI3K and Erk1/2 (see §6.2.1 & 6.2.2).

Stabilization of the glucose-3-transporter (GLUT3)
GLUT3 (SLC2A3), one of 14 glucose transporter family members (GLUT1-14), was originally identified as a neuronal-specific glucose transporter but is also expressed in other tissues with high-energy demands, including cancer cells (Macheda et al., 2005;Thorens & Mueckler, 2010;Cairns et al., 2011).PC was recently found to form a stable complex with GLUT3 in human embryonal carcinoma cells (Tera-1 and NCITT) since GLUT3 can be co-precipitated from cell lysates using PNA or anti-PC antibody (and, inversely, podocalyxin is co-precipitated with anti-GLUT3 antibody) (Schopperle et al., 2010).Suppression of PODXL expression via siRNA also reduced GLUT3 protein concentrations in NCITT (Schopperle et al., 2010) -suggesting that PC may stabilize GLUT3 protein or regulate expression of GLUT3.

Podocalyxin drives tissue morphogenesis and promotes tumour invasion, metastasis and survival
By expressing full-length and mutant forms of PC in cancer cell lines and assessing cell morphology, apical targeting of proteins, motility and invasion, we and others begun to assign specific functions to PC structural domains.An emerging theme is that the highly charged and glycosylated extracellular domain of PC helps to define the apical domain of epithelial sheetsperhaps purely by a biophysical charge-and steric-repulsion mechanism.However, when overexpressed at high concentration, PC can drive epithelial cell morphogenesis and trigger intracellular cytoskeletal rearrangements that support formation of microvilli (possibly as a mechanism for coping with apical domain expansion), reduce adhesion to extracellular matrix and adjacent cells; and, facilitate cell shedding from monolayers.In this way, overexpression of PC promotes EMT-independent tumour invasion and metastasis.Table 3 summarizes these and other tumour-promoting mechanisms regulated by PC.Although the initial apical domain-sorting of PC may be, in part, a property of extracellular or transmembrane determinants, the prevailing evidence suggests that association of PC's intracellular domain with ezrin and NHERF-1/2 (and consequently to F-actin and the cytoskeleton), is responsible for fine-tuning or stabilizing the apical localization of PC in epithelial cells.In other words, NHERF-1/2 and ezrin enhance the efficient and functionally appropriate apical targeting of PC.Subsequently, PC engagement of NHERF-1/2 and ezrin can serve to nucleate signalling complexes at these apical domains and regulate the spatial signaling by these complexes.For example, NHERF-1/2 and ezrin promote activation of Rho-family GTPases, and cooperate to promote the activation of the Erk1/2 and PI3K/Akt pathways.In addition, since NHERF-1/2 and ezrin themselves have multiple proteinprotein interaction domains, regulatory motifs and associate with numerous signalling intermediates (Table 2), the potential function of PC in regulating apical signalling events is likely much more extensive.By recruiting NHERF-1/2 and ezrin, PC has a role in the temporal and spatial localization of signalling intermediates that contribute to enhanced invasion, motility and adhesion-independent survival of tumor cells.PC recruits cortactin, to apical membrane domains by an as-yet undetermined mechanism that may be independent of NHERF-1/2 and ezrin.Cortactin is commonly localized to leading edge tumour cell protrusions, called invadopodia, where it promotes invasive functions, including the secretion of matrix metalloproteinase and regulates cortical actin rearrangements that promote cell morphogenesis and motility.In addition, by stabilizing GLUT3, PC may not only support a metabolic switch to oxygen-poor glycolysis, but also alter downstream glycosylation machinery.Both of these events have the potential to promote adhesion-independent survival and metastasis of tumour cells.Since some sialomucins, including endolyn (CD164) and PSGL-1, have been implicated in enhanced chemokine-mediated migration of hematopoietic cells (Forde et al., 2007;Veerman et al., 2007), we have also examined PC's role in mediating the migration of hematopoietic precursor cells to specific chemokines.Our preliminary work suggests that PC promotes the

Podocalyxin poorly differentiated tumours and promotes EMT and EMTindependent invasion and metastasis in human cancers
Evaluation of PC expression in human cancers, coupled with tumour characteristics and patient outcome data, reveals four cancer-indication "profiles" where high PC expression in primary tumours provides important diagnostic and prognostic information (Table 4 (I-III)) or polymorphisms in PODXL exons predicts cancer risk and tumour aggressiveness (Table 4, IV).These tumour-expression profiles, combined with in vitro and in vivo mechanistic studies, support the contention that PC promotes tumour invasion and metastases.In some human cancers, high PC expression is commonly detected in poorly differentiated, anaplastic tumour subtypes but not in lower grade tumours that retain differentiation markers and characteristics (Profile I).PC expression is also commonly detected in testicular cancer, a germ-line tumour.In these cases, PC may serve as a marker of immature or "dedifferentiated" cells that have undergone EMT or retained a more primitive gene expression profile (perhaps derived from tissue stem cells).However, a causal link between PC expression and tumour progression and invasion is not proven in these profile I cancers.Nevertheless, drawing on in vitro studies, we hypothesize that PC may contribute to (but not initiate) the invasive or aggressive tumour phenotype in some of these cancers.For this reason, cancers of this type may benefit by the advent of PC diagnostics as a supplemental approach to the staging or differential diagnosis of primary tumor origin.We note that the clinical application of PC expression profiling for cancer diagnostics is achievable with existing reagents and technology, requiring only a concerted effort of validation.In some cancers, PC overexpression in rare, highly aggressive tumour subtypes clearly correlates with enhanced tumour invasion, risk of metastasis and poor patient outcome (Profile II).In these cases, we propose that PC expression directly promotes EMTindependent invasion and metastases.We anticipate that further detailed analyses of aggressive tumor sub-types, especially for epithelial carcinomas, will expand the examples of rare, PC expressing, aggressive tumours in profile II.For instance: Because forced expression of PC in a prostate tumour cell line enhances invasive and migration potential, and PC is expressed in malignant prostate tumours (but not primary or normal adjacent tumours) (Fig. 4), we predict that a subset of high PC-expressing primary prostate tumours will display high risk of metastases.Evaluation of PC expression in primary tumours of profile II has the most value for prognostic assessment and the design of treatment and surveillance strategy.Furthermore, we hope that it will be possible to exploit PC expression in these indications for the development of targeted adjuvant therapies (see §7.3).
The detection of PC (or fragments) in serum may also provide a noninvasive biomarker of tumour progression or treatment progress in some cancers (profile III).The case is strongest for testicular cancer where serum PC (aka GCTM-2 or TRA-1-60) may be a more robust marker than current "gold-standard" biomarkers used in testicular cancer diagnostics.We suspect that cancers that commonly express high levels of PC (profile I) may also shed PC fragments into serum and continued research will likely confirm PC's utility as diagnostic serum marker.If so, PC detection in serum may be a generally useful for detecting disease or monitoring tumour progression and treatment efficacy.Finally, genetic profiling of the PODXL locus reveals a fourth profile where polymorphisms in PODXL exons are associated with increase prostate cancer risk and tumor aggressiveness (Table 4 (IV)).Although it will be important to evaluate whether these genetic markers correlate with PC expression in prostate cancer or have any role in PC's functions in these tumours, screening for PODXL polymorphisms has potential theranostic applications in designing treatment strategies for prostate cancer patients.

Podocalyxin-targeted therapies for the treatment of metastatic cancer
There are several challenges to overcome in the development of PC-targeted adjuvant therapies for the treatment of high-risk primary tumours or oncolytic treatment of systemic cancers.First, we do not yet know if the high PC expression detected on primary tumours in the case of aggressive breast, renal cell and ovarian carcinoma is maintained on metastatic tumour cells or following engraftment at a secondary tissue site.However, evaluation of PC expression in breast and ovarian carcinoma cell lines, many of which were derived from metastatic tumours, suggests that PC expression might be a common feature of metastatic breast and ovarian cancer -especially, basal-type breast carcinomas (Fig. 1).Second, any PC-targeted therapies must carefully consider potential renal and vascular toxicity since PC is highly expressed on glomerular podocytes and on most vasculature.Fortunately, there are several examples of uniquely modified, tumour-specific forms of PC.These provide feasible targets for antibody-based drugs that are either directly oncolytic or block PCmediated functions without affecting the function of normal cells.

Fig. 1 .
Fig. 1.Transcript expression levels of PODXL, NHERF1 and NHERF2 in a collection of human breast cancer cell lines.Arrows indicate the luminal-like MCF7 and basal (B)-like MDA.MB.231 breast cancer cell lines commonly used in the study of PC function.The transcript expression heat map was generated from meta-analysis of publically available microarray gene expression profiles of human breast cancer cell lines (Kao et al., 2009) using Gene Cluster 3.0 and Java TreeView.

Fig. 2 .
Fig. 2. Podocalyxin expression defines the apical domain of polarized epithelia but promotes non-EMT loss of adhesion and shedding when overexpressed.Finally, to gain some insight into the in vivo function of PC in the growth, local invasion and metastatic potential of human breast cancers, we have used tumor xenograft models.In preliminary experiments, sub-cutaneous transplantation of MCF7 or PC-overexpressing MCF7 cells into immunodeficient mice (Rag2M) suggests that expression of high levels of

Fig 4 .
Fig 4. Expression of PODXL in normal human prostate tissue and prostate cancer (GEO dataset GSE6919)(Chandran et al., 2007;Yu et al., 2004).The metastatic tumour group is significantly different than the normal adjacent and primary tumour groups but not the normal donor group (*p < 0.05, Tukey one-way ANOVA analysis).All other group comparisons are not significantly different.
& detachment from basement membranes  Inter-and intra-cellular biophysical charge-repulsion  Recruitment/sequestration of cortical actin complexes to apical membrane and weakening of ECM adhesion  Exclusion of integrins from the apical domain Invasion of surrounding tissue  PC association with collagen at leading edge  Recruitment and apical localization of cortactin and induced expression and secretion of MMPs at the leading edge High motility  Enhanced CXCL12/CXCR4 axis signalling and chemotaxis  Activation and localization of Rho-family GTPases  Enhanced activation of PI3K and ERK1/2 Homing and to distant tissue sites  Potential E/L-selectin ligand in some tumours  CXCL12/CXCR4 mediated engraftment and survival in secondary tissue Adherence-independent survival  Enhanced activation of PI3K/Akt and Erk 1/2 pathways  Promote expression/stabilization of GLUT3

Fig. 8 .
Fig. 8. Summary of podocalyxin-mediated molecular mechanisms promoting tumour growth, invasion, migration and adhesion-independent survival.migration of hematopoietic cells and possibly tumor cells to CXCL12, perhaps by stabilizing the surface expression (and localization) of CXCR4.Thus, upregulation of PC on tumors may herald the acquisition of a great capacity for migration to CXCL12 rich tissues including bone, lung and brain.

Profile I .
Podocalyxin is commonly expressed in high-grade or poorly differentiated tumours  Thyroid carcinoma (undifferentiated)  Pancreatic ductal/ampullary carcinoma  Small-cell lung carcinoma  Acute myeloid and lymphoid leukemia and myeloid sarcoma  Anaplastic nephroblastoma  Testicular cancer (germ cell)  Malignant astrocytoma Profile II.Podocalyxin is overexpressed in rare subsets of highly aggressive tumours and predicts poor prognosis  Breast carcinoma  Ovarian carcinoma  Renal cell carcinoma Profile III.PC expression as potential serum marker but clinical significance expression profile is undetermined  Testicular cancer  Colon cancer (cell line) Profile IV.PODXL polymorphisms and SNPs associated with cancer risk and tumor aggressiveness  Prostate carcinoma

Table 4 .
Podocalyxin expression profiles in human cancer.