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CD44

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.

Fibrosis of the lung is characterized by the accumulation of myofibroblasts, a key mediator in the fibrogenic reaction. Cumulative evidence indicates that epithelial-mesenchymal transition (EMT), a process whereby epithelial cells become mesenchyme-like, is an important contributing source for the myofibroblast population. Underlying this phenotypical change is a dramatic alteration in cellular structure. The receptor for advanced glycation end-products (RAGE) has been suggested to maintain lung homeostasis by mediating cell adhesion, while the family of ezrin/radixin/moesin (ERM) proteins, on the other hand, serve as an important cross-linker between the plasma membrane and cytoskeleton. In the present investigation, we tested the hypothesis that RAGE and ERM interact and play a key role in regulating EMT-associated structural changes in alveolar epithelial cells. Exposure of A549 cells to inflammatory cytokines resulted in phosphorylation and redistribution of ERM to the cell periphery and localization with EMT-related actin stress fibers. Simultaneously, blockade of Rho kinase (ROCK) signaling attenuated these cytokine-induced structural changes. Additionally, RAGE expression was diminished after cytokine stimulation, with release of its soluble isoform via a matrix metalloproteinase (MMP)-9-dependent mechanism. Immunofluorescence microscopy and coimmunoprecipitation revealed association between ERM and RAGE under basal conditions, which was disrupted when challenged with inflammatory cytokines, as ERM in its activated state complexed with membrane-linked CD44. Dual-fluorescence immunohistochemistry of patient idiopathic pulmonary fibrosis (IPF) tissues highlighted marked diminution of RAGE in fibrotic samples, together with enhanced levels of CD44 and double-positive cells for CD44 and phospho (p)ERM. These data suggest that dysregulation of the ERM-RAGE complex might be an important step in rearrangement of the actin cytoskeleton during proinflammatory cytokine-induced EMT of human alveolar epithelial cells.

The epithelial-mesenchymal transition (EMT) has been linked to induction of a stem-cell like phenotype, characterized by altered cell surface marker expression and increased tumor formation. The aim of this study was to investigate whether EMT correlates with CD24+CD44+ and CD133+ cells in pancreatic cancer. The morphology of untreated and gemcitabine-treated SW1990 gemcitabine-resistant cells and normal SW1990 cells were compared. NF-kappaB p65 expression was knocked down using siRNA. Vimentin and E-cadherin expression were analyzed using western blotting, and CD24+CD44+, CD133+ cells were quantified by FACS. Additionally, immunohistochemistry of EMT-associated markers and stem cell-associated markers were performed in 41 cases of human pancreatic ductal adenocarcinoma. In SW1990 gemcitabine-resistant cells, gemcitabine induced a mesenchymal cell phenotype, expression of EMT-related molecular markers and increased CD24+CD44+ and CD133+ cells compared to untreated SW1990 gemcitabine-resistant and SW1990 cells. Knockdown of NF-kappaB p65 inhibited the ability of gemcitabine to increase the proportion of CD24+CD44+ or CD133+ cells and expression of EMT-related molecular markers. In human pancreatic ductal adenocarcinoma, significant correlations were observed between expression of the EMT-associated markers vimentin and E-cadherin, and stem cell-associated markers CD24, CD133 and CD44. This study demonstrated that EMT correlated with CD24+CD44+ and CD133+ cells in pancreatic cancer. This study also suggests that EMT may induce cancer stem-like cells in pancreatic cancer, with different degrees of EMT probability inducing different proportions of CD24+CD44+ and CD133+ cells.

Recent studies have shown that cancer stem-like cells (CSCs) within a tumor have the capacity for self-renewal and differentiation, and are associated with an aggressive phenotype and therapeutic resistance. Studies have also associated tumor progression with alterations in the levels of intracellular reactive oxygen species (ROS). In this study, we cultured nasopharyngeal carcinoma (NPC) CSCs in conditions that allowed sphere formation. The resulting sphere cells displayed stemness properties, characteristics of the epithelial-mesenchymal transition (EMT), and increased expression of the CSC surface marker CD44. We further evaluated the association between CD44 expression and EMT marker expression, and any correlation with redox status, in these CSCs. We showed that the EMT in sphere cells is associated with the upregulation of CD44 expression and increased ROS generation, which might promote NPC aggressiveness. We also identified the coexpression of CD44 with the EMT marker N-cadherin in sphere cells, and downregulated CD44 expression after the addition of the antioxidant N-acetyl cysteine. Our results indicate that CD44 plays a role in the EMT phenotype of CSCs in NPC, and suggest its involvement in EMT-associated ROS production. These findings might facilitate the development of a novel therapy for the prevention of NPC recurrence and metastasis.

epithelial-mesenchymal transition (EMT) promotes cancer cell invasion, metastasis and treatment failure. EMT may be activated in cancer cells by reactive oxygen species (ROS). EMT may promote conversion of a subset of cancer cells from a CD44(low)-CD24(high) (CD44L) epithelial phenotype to a CD44(high)-CD24(-/low) (CD44H) mesenchymal phenotype, the latter associated with increased malignant properties of cancer cells. ROS are required for cells undergoing EMT, although excessive ROS may induce cell death or senescence; however, little is known as to how cellular antioxidant capabilities may be regulated during EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigate mechanisms of SOD2 transcriptional regulation in EMT, as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference and flow cytometric approaches, we find that transforming growth factor (TGF)-beta stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed keratinocytes was concurrent with suppression of TGF-beta-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-kappaB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. These data provide novel mechanistic insights into the dynamic expression of SOD2 during EMT. In addition, we delineate a functional role for SOD2 in EMT via the influence of this antioxidant upon distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology.

The alteration of epithelial stability, which includes changes in the expression of E-cadherin and CD44, is one of the complex biomolecular mechanisms involved in the tumoral epithelial-mesenchymal transition (EMT) process. We followed the E-cadherin/CD44 immunophenotype by single and double detections in 25 cases of bladder urothelial carcinomas. Our study investigated simultaneously the differences in expression of the two markers, in different tumoral compartments, according to the prognostic parameters of the lesions. The study indicated significant differences in the expression of E-cadherin in relation to tumor grade, depth of invasion, tumor stage and Ki-67 proliferation index (PI), both intratumoral and at the advancing edge. For CD44, expression differences were found between the tumor grades in intratumoral sites, while for both intratumoral and advancing edge compartments the differences occurred for the depth of tumor invasion, tumor stage and Ki-67 PI. The only differences in the expression of the two markers in relation with the presence of lymph node metastasis were for E-cadherin at the advancing edge. In this study, the intratumoral E-caderin-/CD44- immunophenotype, respectively E-caderin-/CD44+ at the advancing edge were associated with the tumor aggressiveness analyzed parameters. The maintenance of CD44 expression at the advancing edge represents a negative prognostic factor for bladder urothelial carcinoma and supports the implication of EMT process, through the existence at this level of a cell population with particular properties.

The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial-mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S-HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions.CI - Copyright (c) 2015. Published by Elsevier B.V.

cancer metastasis is strongly correlated with epithelial-mesenchymal transition (EMT), in which transforming growth factor-beta (TGF-beta) signaling plays a central role. CD44 has emerged as a cancer stem cell (CSC) marker that strongly induces EMT together with TGF-beta1. This study aimed to investigate the link between high CD44 and TGF-beta1 levels during EMT in HCC cell lines. FACS analysis showed high expression of CD44 in TGF-beta1-positive SNU-368 cells and TGF-beta1-negative SNU-354 cells. SNU-368 CD44(+) cells showed EMT through up-regulation of the AKT/GSK-3beta/beta-catenin pathway. By comparison, SNU-354 CD44(+) cells showed only increased N-cadherin expression, which was not accompanied by a decrease in E-cadherin expression, and also down-regulated the AKT/GSK-3beta/beta-catenin pathway. However, TGF-beta1-stimulated SNU-354 cells (CD44/TGF-beta1(+)) exhibited lower E-cadherin and higher N-cadherin expression with increased AKT/GSK-3beta/beta-catenin pathway activity. CD44/TGF-beta1(+) SNU-354 cells also showed enhanced migration and formed larger spheres, while the TGF-beta1-induced stem cell properties returned to their original state with the TGF-beta1 inhibitor SB431542. SB431542-treated SNU-368 (CD44/TGF-beta1(-)) cells also showed diminished N-cadherin and AKT/GSK-3beta/beta-catenin pathway activity and further decreased cell motility in a wound healing assay. However, CD44 knockdown in SNU-354 cells did not induce EMT even after treatment with TGF-beta1. Finally, double inhibition of both CD44 and TGF-beta1 further decreased migration and sphere formation more strongly than a single inhibition in SNU-368 cells. In conclusion, the current study demonstrated the synergistic interactions between CD44 and TGF-beta1 in EMT induction and CSC properties through the AKT/GSK-3beta/beta-catenin pathway in HCC cells.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

Increased expression of zinc finger E-box binding homeobox 1 (ZEB1) is associated with tumor grade and metastasis in lung cancer, likely due to its role as a transcription factor in epithelial-to-mesenchymal transition (EMT). Here, we modeled malignant transformation in human bronchial epithelial cells (HBECs) and determined that EMT and ZEB1 expression are early, critical events in lung cancer pathogenesis. Specific oncogenic mutations in TP53 and KRAS were required for HBECs to engage EMT machinery in response to microenvironmental (serum/TGF-beta) or oncogenetic (MYC) factors. Both TGF-beta- and MYC-induced EMT required ZEB1, but engaged distinct TGF-beta-dependent and vitamin D receptor-dependent (VDR-dependent) pathways, respectively. Functionally, we found that ZEB1 causally promotes malignant progression of HBECs and tumorigenicity, invasion, and metastases in non-small cell lung cancer (NSCLC) lines. Mechanistically, ZEB1 expression in HBECs directly repressed epithelial splicing regulatory protein 1 (ESRP1), leading to increased expression of a mesenchymal splice variant of CD44 and a more invasive phenotype. In addition, ZEB1 expression in early stage IB primary NSCLC correlated with tumor-node-metastasis stage. These findings indicate that ZEB1-induced EMT and associated molecular changes in ESRP1 and CD44 contribute to early pathogenesis and metastatic potential in established lung cancer. Moreover, TGF-beta and VDR signaling and CD44 splicing pathways associated with ZEB1 are potential EMT chemoprevention and therapeutic targets in NSCLC.

Cell surface proteins such as CD44 and CD24 are used to distinguish cancer stem cells (CSCs) from the bulk-tumor population. However, the molecular functionalities of CD24 and CD44, and how these two molecules coordinate in CSCs remain poorly understood. We found that nasopharyngeal carcinoma (NPC) cells with high expression of CD44 and CD24 proteins presented with pronounced CSC properties. Accordingly, a subpopulation of NPC cells with co-expression of CD44 and CD24 were specially enriched in high-stage clinical samples. Furthermore, ectopically expressing the epithelial-mesenchymal transition (EMT) regulator Twist was able to upregulate the stemness factors, and vice versa. This indicates a reciprocal regulation of stemness and EMT. Intriguingly, we found that this reciprocal regulation was differentially orchestrated by CD44 and CD24, and only simultaneous silencing the expression of CD44 and CD24 led to a broad-spectrum suppression of CSC properties. Oppositely, overexpression of CD44 and CD24 induced the reprogramming of parental NPC cells into CSCs through STAT3 activation, which could be blunted by STAT3 inhibition, indicating that CD44 and CD24 collaboratively drive the reprogramming of NPC cells through STAT3-mediated stemness and EMT activation. Consequently, targeting of the CD44/CD24/STAT3 axis may provide a potential therapeutic paradigm for the treatment of NPC through repressing CSC activities.

CD44, a major surface receptor for hyaluronic acid, has multiple isoforms and represents a major cancer stem cell marker for various epithelial tumors. CD44 variant 9 (CD44v9) was correlated with recurrence and metastasis in gastric and colon cancer. We examined its role in invasion and as a biomarker for the basal muscle invasive molecular subtype showing worse prognosis, and for tumor progression in high risk (pT1/high grade) nonmuscle invasive bladder cancers (NMIBCs). CD44v9, cytokeratin 5/6 (CK5/6), and cytokeratin 20 (CK20) expression was evaluated by immunohistochemistry in 98 pathologically confirmed specimens (36 muscle and 62 highrisk nonmuscle) and correlated to clinical outcome. In vitro analysis was performed using two human bladder cancer cell lines (HT1376 and 5637). The CD44v9 highexpressing group exhibited significantly lower progressionfree and cancerspecific survival rates in both muscle (P=0.0349 and 0.0382, respectively) and nonmuscle (P=0.0002 and 0.0079) invasive patients. CD44v9 expression was significantly correlated with CK5/6 (P<0.001), colocalizing at the muscle invasion front but distinctly separated from CK20 in nonmuscle invasion. CD44 and CD44v9 siRNA knockdown demonstrated significantly lower Matrigel invasion ability and significantly shorter migration distance (all P<0.01). CD44 and CD44v9 knockdown increased Ecadherin and decreased Ncadherin, snail, and slug epithelialmesenchymal transition marker protein expression. Thus, higher CD44v9 expression was associated with worse prognosis, likely impacting invasion and migration via the epithelialmesenchymal transition. Together, these findings suggest that CD44v9 expression might be a useful predictive biomarker in basaltype muscle and high-risk NMIBC.

BACKGROUND: epithelial-mesenchymal transition is one of the mechanisms that contribute to the aggressiveness of cancer. CD44 antigen is an emerging biomarker that is currently being evaluated in the literature in the frame of epithelial-mesenchymal transition. The aim of this study was to evaluate the expression of CD44 in relation to several clinicopathological parameters in gastric and esophageal carcinomas. MATERIALS AND METHODS: This historical cohort survey was performed on gastric and esophageal tumors obtained from 86 patients who underwent resection from 2003 until 2011. Immunohistochemistry was used for assessing the expression of CD44 with a semi-quantitative model. RESULTS: Survival rates were negatively correlated with pT at diagnosis (p<0.01), pN (p<0.01), positive lymph node ratio (p<0.01), pM (p=0.004), tumor stage (p<0.01) and grade (p<0.01), and the absence of R0 resection (p<0.01). disease-free survival rates were affected by pN at diagnosis (p=0.02), positive lymph node ratio (p=0.03) and tumor stage (p=0.02). The correlation of tissue CD44 expression with overall and disease-free survival was not statistically significant, whereas tumor T, N, positive lymph node ratio and lymphovascular invasion were significantly associated with CD44 positivity. Moreover, tissue CD44 expression was not significantly associated with stage, histological subtype according to Lauren classification, the patient's age or gender. CONCLUSION: CD44 antigen expression might be an indicative and descriptive marker of epithelial-mesenchymal transition, mainly in gastric cancer, reflecting diverse clinicopathological events.CI - Copyright (c) 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Gallbladder cancer (GBC) is one of the most unfavorable prognostic tumor, and immediate growth and distant metastasis are important factors associated with the poor prognosis of patients with this disease. Standard and variant isoforms of CD44 are associated with tumor growth, metastasis, and epithelial-mesenchymal transition (EMT), although their roles in GBC are unclear. We investigated the relationship between the CD44 isoforms with EMT, chemotaxis, and tumorigenicity. We analyzed CD44 expression in the GBC cell line NOZ and found that it comprises a major population that expressed CD44std+/CD44v9- (CD44s) and the minor population that expressed CD44std-/CD44v9+ (CD44v). CD44s cells exhibited increased chemotaxis and invasiveness compared with CD44v cells in in vitro cell migration and invasion assays. CD44s cells expressed higher and lower levels of mRNAs that encode vimentin and E-cadherin, respectively, compared with those of CD44v cells. CD44s cells expressed high levels of the transcription factors ZEB1 and ZEB2 that mediate EMT, and low levels of a splicing factor ESRP1 that controls the CD44 isoform switch. We performed in vivo mouse xenotransplantation analyses of CD44s and CD44v cells and found that CD44v cells exhibited relatively increased tumorigenicity. Immunohistochemical analysis of tissue microarrays revealed that high levels of CD44v9 and CD44std were associated with poorer prognosis. The expression of CD44std was also associated with poorly differentiated tumors and distant metastasis. In conclusion, CD44s was associated with a mesenchymal phenotype, increased chemotaxis and invasiveness, and decreased tumorigenicity. In contrast, CD44v cells exhibited an epithelial phenotype, decreased chemotaxis, decreased invasiveness, and increased tumorigenicity. These findings suggest that CD44v and CD44s cells play differently important roles in the progression and metastasis of GBC and the isoform switch triggers EMT.

Increased metastasis and a precipitous recurrence contribute to the lethality of ovarian cancer (OC). Several molecular mechanisms including aberrant-splicing have been closely associated with the extent of cancer progression. Numerous gene transcripts are differentially spliced in cancer cells, CD44 being one of them. CD44 splice isoforms contribute to the aggressiveness and gain of stem-like properties in different cancer types, but their role in ovarian cancer remains to be elucidated. We observed augmented CD44 levels in human ovarian cancer patient samples correlated with enhanced expression of the mesenchymal spliced variant CD44s (standard) and a concurrent decrease in the epithelial variants (CD44v). Moreover, CD44s was upregulated upon TGFbeta1-induced EMT, which was mediated through the downregulation of the splicing factor, ESRP1. Furthermore, overexpression of this mesenchymal isoform in the OC cells induced EMT and invasion, followed by the gain of stem-like characteristics and chemoresistance. Since all these phenomena render lethality to this disease type, CD44s can be attributed for playing a major role in deregulated-splicing mediated ovarian cancer progression.CI - (c) 2017 Wiley Periodicals, Inc.

Disseminated cancer cells in malignant ascites possess unique properties that differ from primary tumors. However, the biological features of ascites tumor cells (ATC) have not been fully investigated. By analyzing ascites fluid from 65 gastrointestinal cancer patients, the distinguishing characteristics of ATC were identified. High frequency of CD44(+) cells was observed in ATC using flow cytometry (n = 48). Multiplex quantitative PCR (n = 15) showed higher gene expression of epithelial-mesenchymal transition (EMT)-related genes and transforming growth factor beta (TGF-beta)-related genes in ATC than in the primary tissues. Immunohistochemistry (n = 10) showed that ATC also had much higher expression of phosphorylated SMAD2 than that in the corresponding primary tissues. TGF-beta 1 was detected in all cases of malignant ascites by enzyme-linked immunoassay (n = 38), suggesting the possible interaction of ATC and the ascites microenvironment. In vitro experiments revealed that these ATC properties were maintained by TGF-beta 1 in cultured ATC(n = 3). Here, we showed that ATCrevealed high frequencies of CD44 and possessed distinct EMT features from primary tissues that were mainly maintained by TGF-beta 1 in the ascites.CI - (c) 2018 The Authors. cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese cancer Association.

Gastric cancer is a common malignancy worldwide. The prognosis of early stage gastric cancer patients has significantly improved in recent years. However, in progressive stage gastric cancer patients, the prognosis remains relatively poor due to tumor metastases. In our previous study, we showed that the expression of miR711 in gastric cancer tissues is low, and restoration of miR711 inhibited the invasion and migration and the occurrence of epithelialmesenchymal transition (EMT) in gastric cancer cells. Yet, the mechanisms involved in these processes remain unknown. In the present study, we demonstrated that miR711mediated downregulation of CD44 expression inhibited EMT of gastric cancer cells in vitro and in vivo by downregulating vimentin protein expression and upregulating Ecadherin protein expression through transfection, qRTPCR and western blotting. Therefore, miR711 may provide a promising target for EMTrelated therapy for gastric cancer.