Podoplanin is a small membrane mucin expressed in tumors associated with malignant progression. It is enriched at cell-surface protrusions where it colocalizes with members of the ERM (ezrin, radixin, moesin) protein family. Here, we found that human podoplanin directly interacts with ezrin (and moesin) in vitro and in vivo through a cluster of basic amino acids within its cytoplasmic tail, mainly through a juxtamembrane dipeptide RK. Podoplanin induced an epithelial-mesenchymal transition in MDCK cells linked to the activation of RhoA and increased cell migration and invasiveness. Fluorescence time-lapse video observations in migrating cells indicate that podoplanin might be involved in ruffling activity as well as in retractive processes. By using mutant podoplanin constructs fused to green fluorescent protein we show that association of the cytoplasmic tail with ERM proteins is required for upregulation of RhoA activity and epithelial-mesenchymal transition. Furthermore, expression of either a dominant-negative truncated variant of ezrin or a dominant-negative mutant form of RhoA blocked podoplanin-induced RhoA activation and epithelial-mesenchymal transition. These results provide a mechanistic basis to understand the role of podoplanin in cell migration or invasiveness.

We report the full cDNA sequence encoding the human homologue of murine PA2.26 (T1α‐2, podoplanin), a small mucin‐type transmembrane glycoprotein originally identified as a cell‐surface antigen induced in keratinocytes during mouse skin carcinogenesis. The human PA2.26 gene is expressed as 2 transcripts of 0.9 and 2.7 kb in several normal tissues, such as the placenta, skeletal muscle, heart and lung. Using a specific polyclonal antibody raised against a synthetic peptide of the protein ectodomain, PA2.26 was immunohistochemically detected in about 25% (15/61) of human early oral squamous cell carcinomas. PA2.26 distribution in the tumours was heterogeneous and often restricted to the invasive front. Double immunofluorescence and confocal microscopy analysis showed that PA2.26 colocalized with the membrane cytoskeleton linker ezrin at the surface of tumour cells and that its presence in vivo was associated with downregulation of membrane E‐cadherin protein expression. Ectopic expression of human PA2.26 in HeLa carcinoma cells and immortalized HaCaT keratinocytes promoted a redistribution of ezrin to the cell edges, the formation of cell‐surface protrusions and reduced Ca²⁺‐dependent cell‐cell adhesiveness. These results point to PA2.26 as a novel biomarker for oral squamous cell carcinomas that might be involved in migration/invasion.

Podoplanin is a small cell-surface mucin-like glycoprotein that plays a crucial role in the development of the alveoli, heart, and lymphatic vascular system. Emerging evidence indicates that it is also involved in the control of mammary stem-cell activity and biogenesis of platelets in the bone marrow, and exerts an important function in the immune response. Podoplanin expression is upregulated in different cell types, including fibroblasts, macrophages, T helper cells, and epithelial cells, during inflammation and cancer, where it plays important roles. Podoplanin is implicated in chronic inflammatory diseases, such as psoriasis, multiple sclerosis, and rheumatoid arthritis, promotes inflammation-driven and cancer-associated thrombosis, and stimulates cancer cell invasion and metastasis through a variety of strategies. To accomplish its biological functions, podoplanin must interact with other proteins located in the same cell or in neighbor cells. The binding of podoplanin to its ligands leads to modulation of signaling pathways that regulate proliferation, contractility, migration, epithelial–mesenchymal transition, and remodeling of the extracellular matrix. In this review, we describe the diverse roles of podoplanin in inflammation and cancer, depict the protein ligands of podoplanin identified so far, and discuss the mechanistic basis for the involvement of podoplanin in all these processes.

Pharmacological activation of cannabinoid receptors elicits antitumoral responses in different cancer models. However, the biological role of these receptors in tumor physio-pathology is still unknown. We analyzed CB₂ cannabinoid receptor protein expression in two series of 166 and 483 breast tumor samples operated in the University Hospitals of Kiel, Tübingen, and Freiburg between 1997 and 2010 and CB₂ mRNA expression in previously published DNA microarray datasets. The role of CB₂ in oncogenesis was studied by generating a mouse line that expresses the human V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2) rat ortholog (neu) and lacks CB₂ and by a variety of biochemical and cell biology approaches in human breast cancer cells in culture and in vivo, upon modulation of CB₂ expression by si/shRNAs and overexpression plasmids. CB₂-HER2 molecular interaction was studied by colocalization, coimmunoprecipitation, and proximity ligation assays. Statistical tests were two-sided. We show an association between elevated CB₂ expression in HER2+ breast tumors and poor patient prognosis (decreased overall survival, hazard ratio [HR] = 0.29, 95% confidence interval [CI] = 0.09 to 0.71, P = .009) and higher probability to suffer local recurrence (HR = 0.09, 95% CI = 0.049 to 0.54, P = .003) and to develop distant metastases (HR = 0.33, 95% CI = 0.13 to 0.75, P = .009). We also demonstrate that genetic inactivation of CB₂ impairs tumor generation and progression in MMTV-neu mice. Moreover, we show that HER2 upregulates CB₂ expression by activating the transcription factor ELK1 via the ERK cascade and that an increased CB₂ expression activates the HER2 pro-oncogenic signaling at the level of the tyrosine kinase c-SRC. Finally, we show HER2 and CB₂ form heteromers in cancer cells. Our findings reveal an unprecedented role of CB₂ as a pivotal regulator of HER2 pro-oncogenic signaling in breast cancer, and they suggest that CB₂ may be a biomarker with prognostic value in these tumors.

Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalize at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin–CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.

Vitamin D receptor (VDR) mediates the antitumoral action of the active vitamin D metabolite 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). VDR expression is lost during colon cancer progression causing unresponsiveness to 1,25(OH)₂D₃ and its analogs. Previously, Snail1, an inducer of epithelial-to-mesenchymal transition (EMT), was reported to inhibit VDR expression. Here, we show that Snail2/Slug, but not other EMT inducers such as Zeb1, Zeb2, E47 or Twist1, represses VDR gene promoter. Moreover, Snail2 and Snail1 show additive repressing effect on VDR promoter. Snail2 inhibits VDR RNA and protein and blocks the induction of E-cadherin and an adhesive phenotype by 1,25(OH)₂D₃. Snail2 reduces the ligand-induced VDR transcriptional activation of a consensus response element and of the CYP24 promoter. Concordantly, Snail2 inhibits the induction of CYP24 RNA and p21^CIP1, filamin A and vinculin proteins and the repression of c-MYC by 1,25(OH)₂D₃. Additionally, Snail2 abrogates β-catenin nuclear export and the antagonism of the transcriptional activity of β-catenin–T-cell factor complexes by 1,25(OH)₂D₃. SNAI2 expression is upregulated in 58% of colorectal tumors and correlates inversely with that of VDR. However, VDR downregulation is higher in tumors coexpressing SNAI2 and SNAI1 than in those expressing only one of these genes. Together, these data indicate that Snail2 and Snail1 cooperate for VDR repression in colon cancer.