Prognostic effect of programmed cell death ligand 1/programmed cell death 1 expression in cancer stem cells of human oral squamous cell carcinoma.

The relationship between cancer stem cells (CSCs) in oral squamous cell carcinoma (OSCC) and programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) remains unclear. Therefore, the present study aimed to clarify the association between the CD44v3high/CD24low immunophenotype of CSCs in OSCC and PD-L1/PD-1 co-expression, and to assess the prognostic effect of CSCs in terms of immune checkpoint molecules. Formalin-fixed, paraffin-embedded tissue samples and clinicopathological data from 168 patients with OSCC were retrospectively retrieved. Immunohistochemical staining and reverse transcription quantitative polymerase chain reaction were applied to a tissue microarray of the invasive front of each case. Semi-automated cell counting was used to assess CD44v3, CD24, PD-L1 and PD-1 expression by immunohistochemistry (IHC) using a digital image analysis program. Associations between immunological markers and clinicopathological variables were estimated. Patients with the CSC immunophenotype CD44v3high/CD24low, and patients with a high PD-L1/PD-1-positive cell density in the tumor parenchyma and stroma had significantly lower survival rates. Furthermore, patients with the CSC immunophenotype (CD44v3high/CD24low) and high PD-L1/PD-1 co-expression had even lower survival rates (P<0.01, log-rank test). Notably, there was a positive correlation between CD44v3 and PD-L1 expression (τ=0.1096, P=0.0366, Kendall rank correlation coefficient) and a negative correlation between CD24 and PD-1 expression (τ=-0.1387, P=0.0089, Kendall rank correlation coefficient). Additionally, the high CD44v3 expression group, as determined by IHC, exhibited significantly decreased expression of U2 small nuclear RNA auxiliary factor 1 (U2AF1) at the mRNA level compared with that in the low CD44v3 expression group (P<0.001, Mann-Whitney U test), and U2AF1 and PD-L1 mRNA expression exhibited a significant negative correlation (τ=-0.3948, P<0.001, Kendall rank correlation coefficient). In conclusion, CSCs in OSCC may evade host immune mechanisms and maintain CSC stemness via PD-L1/PD-1 co-expression, resulting in unfavorable clinical outcomes.

Decreased CD44v3 expression impairs endometrial stromal cell proliferation and decidualization in women with recurrent implantation failure.

BACKGROUND: The precise pathogenesis of poor endometrial receptivity in recurrent implantation failure (RIF) remains unclear. This study was aimed at exploring the effects of different CD44 isoforms in the mid-secretory phase endometrium on endometrial receptivity in women with RIF. METHODS: Mid-secretory phase endometrial tissue samples were obtained from the following two groups of women who had undergone IVF: (a) 24 patients with RIF and (b) 18 patients with infertility due to tubal obstruction, who had achieved a successful clinical pregnancy after the first embryo transfer in IVF (control group). Identification of differentially expressed CD44 isoforms in endometrial tissues was assessed using immunohistochemistry, qPCR, and western blotting. Effects of overexpression and knockdown of CD44v3 on proliferation and decidualization of immortalized human endometrial stromal cells (T-HESCs) and primary HESCs were investigated by qPCR and western blot analysis. A heterologous coculture system of embryo implantation was constructed to mimic the process of trophoblast invasion during implantation. RESULTS: The expression of CD44v3 was significantly higher in the mid-secretory phase of endometrial stromal cells than in the proliferation phase, but was notably lower in RIF patients. Knockdown of CD44v3 significantly downregulated cell proliferation both in T-HESCs and HESCs. The expression of decidualization markers, prolactin (PRL) and insulin like growth factor binding protein-1 (IGFBP1), was notably decreased following the knockdown of CD44v3, whereas the expression of both PRL and IGFBP1 increased after its overexpression in HESCs. Furthermore, the CD44v3-knockdown HESCs displayed significant deficiency in supporting trophoblast outgrowth in a coculture system of embryo implantation; however, overexpression of CD44v3 in HESCs promoted trophoblast outgrowth. CONCLUSION: The reduced expression of CD44v3 suppresses the proliferation and decidualization of HESCs, which might play a pivotal role in poor endometrial receptivity in women with RIF.

CD44V3, an Alternatively Spliced Form of CD44, Promotes Pancreatic Cancer Progression.

Pancreatic cancer is one of the most lethal malignant tumors. However, the molecular mechanisms responsible for its progression are little known. This study aimed to understand the regulatory role of CD44V3 in pancreatic cancer. A Kaplan-Meier analysis was performed to reveal the correlation between CD44/CD44V3 expression and the prognosis of pancreatic cancer patients. CD44V3 and U2AF1 were knocked down using shRNAs. The proliferation, migration, invasion, and stemness of two pancreatic cell lines, BxPC-3 and AsPC-1, were examined. The expression of CD44V3, cancer-associated markers, and the activation of AKT signaling were detected by qRT-PCR and Western blot. Both CD44 and CD44V3 expression levels were associated with a poor prognosis in pancreatic cancer patients. Interestingly, the expression of CD44V3, instead of CD44, was greatly increased in tumor tissues. CD44V3 knockdown inhibited the proliferation, migration, invasion, and stemness of cancer cells. CD44V3 splicing was regulated by U2AF1 and downregulation of U2AF1 enhanced CD44V3 expression, which promoted pancreatic cancer progression. CD44V3 is an important cancer-promoting factor, which may serve as a potential candidate for pancreatic cancer intervention.

CD44v3,8-10 is essential for Slug-dependent vimentin gene expression to acquire TGF-ß1-induced tumor cell motility.

CD44 is a widely expressed polymorphic adhesion molecule that has pleiotropic functions in development and tumor progression. Its mRNA undergoes alternative splicing to generate multiple variant (CD44v) isoforms, although the function of each CD44v isoform is not fully elucidated. Here, we show that CD44v plays an important role in the induction of vimentin expression upon transforming growth factor-ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT). Among multiple CD44v isoforms expressed in NUGC3 gastric cancer cells, CD44v8-10 and CD44v3,8-10 are involved in the acquisition of migratory and invasive properties associated with TGF-ß1-induced EMT, and only CD44v3,8-10 induces the transcription of vimentin mediated by the EMT transcription factor Slug. In primary tumor specimens obtained from patients with gastric cancer, CD44-containing variant exon 9 (CD44v9) expression and EMT features [E-cadherin(-)vimentin(+)] were significantly correlated, and EMT features in the cells expressing CD44v9 were associated with tumor invasion depth, lymph node metastasis, and pStage, which indicate invasive and metastatic properties, and poor prognosis. These results indicate that certain CD44v isoforms promote tumor cell motility and metastasis in gastric cancer in association with EMT features, and CD44v3,8-10 may contribute to these clinical characteristics.

Direct interaction of TrkA/CD44v3 is essential for NGF-promoted aggressiveness of breast cancer cells.

BACKGROUND: CD44 is a multifunctional membrane glycoprotein. Through its heparan sulfate chain, CD44 presents growth factors to their receptors. We have shown that CD44 and Tropomyosin kinase A (TrkA) form a complex following nerve growth factor (NGF) induction. Our study aimed to understand how CD44 and TrkA interact and the consequences of inhibiting this interaction regarding the pro-tumoral effect of NGF in breast cancer. METHODS: After determining which CD44 isoforms (variants) are involved in forming the TrkA/CD44 complex using proximity ligation assays, we investigated the molecular determinants of this interaction. By molecular modeling, we isolated the amino acids involved and confirmed their involvement using mutations. A CD44v3 mimetic peptide was then synthesized to block the TrkA/CD44v3 interaction. The effects of this peptide on the growth, migration and invasion of xenografted triple-negative breast cancer cells were assessed. Finally, we investigated the correlations between the expression of the TrkA/CD44v3 complex in tumors and histo-pronostic parameters. RESULTS: We demonstrated that isoform v3 (CD44v3), but not v6, binds to TrkA in response to NGF stimulation. The final 10 amino acids of exon v3 and the TrkA H112 residue are necessary for the association of CD44v3 with TrkA. Functionally, the CD44v3 mimetic peptide impairs not only NGF-induced RhoA activation, clonogenicity, and migration/invasion of breast cancer cells in vitro but also tumor growth and metastasis in a xenograft mouse model. We also detected TrkA/CD44v3 only in cancerous cells, not in normal adjacent tissues. CONCLUSION: Collectively, our results suggest that blocking the CD44v3/TrkA interaction can be a new therapeutic option for triple-negative breast cancers.

1,2,3,4,6-Penta-O-galloyl-ß-D-glucose Inhibits CD44v3, a cancer stem cell marker, by regulating its transcription factor, in human pancreatic cancer cell line.

Inhibition of cluster of differentiation 44 (CD44), a pancreatic cancer stem cell (CSC) marker, is a potential treatment for pancreatic ductal adenocarcinoma (PDAC). In this study, we evaluated the effect of 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG), a gallotannin contained in various medicinal plants, on CD44 standard (CD44s) and CD44 variant 3 (CD44v3) in Mia-PaCa-2, human pancreatic cancer cells and explored the underlying mechanisms. PGG showed cytotoxic effects and inhibited the proliferation of Mia-PaCa-2 cells. It also inhibited clonogenic activity, adhesion to fibronectin, and cell migration, which are characteristics of CSCs. PGG inhibited the expression of CD44s and CD44v3 by inducing the phosphorylation of p53 and suppressing NF-κB and Foxo3. Inhibition of Foxo3 induces CD44v3 ubiquitination. Indeed, PGG increased proteasome activity and promoted CD44v3 ubiquitination. PGG downregulated the CSC regulatory factors Nanog, Oct-4, and Sox-2, which act downstream of CD44v3 signaling. These data indicate that PGG may have therapeutic effects in pancreatic cancer mediated by inhibition of CSC markers.

Downregulation of Hyaluronic acid-CD44 signaling pathway in cervical cancer cell by natural polyphenols Plumbagin, Pongapin and Karanjin.

Hyaluronic acid (HA)-CD44 pathway showed association with several malignancies. The natural polyphenols Plumbagin, Pongapin and Karanjin showed anti-cancer activities in different tumors including cervical carcinoma. To understand their mechanism of anti-cancer activity, the effect of the compounds on HA-CD44 pathway was analyzed in cervical cancer cell line HeLa. The mRNA expression of three different isoforms of CD44 i.e., CD44s, CD44v3, and CD44v6, was differentially downregulated by the compounds. This was validated by Western blot and immunocytochemical analysis of CD44s.The low molecular weight HA (LMW-HA) showed growth promoting activity in HeLa at low concentration, whereas high molecular weight HA (HMW-HA) had no such effect. The compounds could preferentially downregulate the LMW-HA level in HeLa, as evident in the cell as well as in the cell-free conditioned medium. Concentration-dependent upregulation of HA synthase-2 (HAS2) was seen in the cell by the compounds, whereas differential downregulation of hyalurinidases 1-4 (HYAL 1-4), predominantly HYAL1, were seen. The compounds could also downregulate the downstream target of the pathway p-AKT (T-308) in concentration-dependent manner. Thus, the compounds could attenuate the HA-CD44 pathway in HeLa cell to restrict the tumor growth.

CD44v3 protein-carrying tumor-derived exosomes in HNSCC patients' plasma as potential noninvasive biomarkers of disease activity.

The molecular cargo of tumor-cell-derived exosomes (TEX) mimics that of parental tumor cells. Thus, TEX could potentially serve as noninvasive biomarkers of cancer progression. However, separation of TEX from non-TEX in patients' plasma requires tumor antigen-specific detection reagents. CD44v3 has been of interest as a potential biomarker of disease progression in HNSCC, because its overexpression in tumor cells associates with poor outcome. Here, CD44v3+ TEX immunocaptured from plasma of 44 HNSCC patients and 7 healthy donors (HDs) were evaluated as potential biomarkers of disease activity and stage. Exosomes were isolated from plasma of by size exclusion chromatography. Using anti-CD44v3 or anti-CD3 mAbs on beads, CD44v3+ TEX CD3(-)TEX-enriched exosomes were immunocaptured from supernatants of nonmalignant or HNSCC cell lines and from patients' plasma. On-bead flow cytometry was used for the detection of FAS-L, PD-L1, TGFF-ß. CSPG4 or EGFR on exosomes. The TEX expression profiles were correlated to clinicopathological parameters. Relative florescence intensity (RFI) values for CD44v3 were higher (p < .01) on TEX from HNSCC cell lines or on CD44v3+ CD3(-) plasma-derived exosomes. RFI values of CD44v3 on CD3(-) exosomes were higher (p < .005) in patients than in HDs and correlated (p < .05) with the UICC stage and lymph node metastasis. In HNSCC patients, CD44v3+ exosomes higher levels of immunosuppressive proteins compared to CD44v3(-) exosomes (p < .05-p < .005), and RFI values for these markers correlated with higher disease stages and lymph node metastasis. Isolation of CD44v3+ exosomes by immunocapture allowed for enrichment of TEX which are potentially promising liquid biomarkers of the tumor burden and disease stage in HNSCC.

CD44v3 and CD44v6 isoforms on T cells are able to discriminate different disease activity degrees and phenotypes in systemic lupus erythematosus patients.

BACKGROUND: Adhesion molecule CD44 contributes to T cell migration into target organs. A higher expression of CD44v3 and v6 isoforms has been identified on T cells from systemic lupus erythematosus (SLE) patients. The aim of this study was to investigate the expression of CD44v3/v6 on T cells of SLE patients in order to evaluate their correlation with clinical features. METHODS: Sixteen healthy subjects (HSs) and 33 SLE female patients were enrolled. Fifteen patients were in remission (Systemic Lupus Erythematosus Disease Activity Index-2000 (SLEDAI-2K) = 0) and 18 patients had an active disease (SLEDAI-2K ≥ 4). Experiments were conducted by flow cytometry. RESULTS: Expression of CD44v3 on CD4+ and CD8+ T cells was higher in active patients compared to HSs ( p = 0.0097 and p = 0.0096). CD44v3 on CD8+ T cells was also higher in active patients compared to patients in remission ( p = 0.038). CD44v6 was higher on CD4+ and CD8+ T cells from active patients compared to HSs ( p = 0.003 and p = 0.0036) and to patients in remission ( p = 0.01 and p = 0.02). In active patients the ratio CD44v3/v6 was unbalanced towards isoform v6 on both T cell populations. In a receiver operating characteristic curve analysis, CD44v6 on CD4+ T cells was the most sensitive and specific one (specificity of 81.8%, sensitivity of 75%). Expression of CD44v6 on CD4+ and CD8+ T cells correlated with the SLEDAI-2K ( p = 0.03, r = 0.38 and p = 0.02, r = 0.39). CD44v6 and CD44v3 on CD8+ T cells associated with nephritis and arthritis ( p = 0.047 and p = 0.023). CONCLUSIONS: CD44v3/v6 can be used as biomarkers of disease activity and phenotypes; isoform v6 on CD4+ T cells can be useful as a diagnostic biomarker.

Development of a sandwich enzyme-linked immunosorbent assay for the detection of CD44v3 using exon v3- and v6-specific monoclonal antibody pairs.

It has been suggested that soluble CD44 levels in cancer patient sera may be closely associated with tumor progression and metastasis. However, to date, there has been limited methodology for detecting the soluble CD44 variant 3 isoform (CD44v3). Herein, using phage display technology, we isolated monoclonal antibodies specific to exon v3 or v6 of CD44 (CD44-exonv3 or CD44-exonv6) from a human synthetic antibody library. We also confirmed the specificity of antibody binding to CD44-exonv3 or -exonv6. Label-free kinetic analysis using the Octet biolayer interferometry system showed that the Kd values of the anti-CD44-exonv3 and anti-CD44-exonv6 antibodies for CD44v3-10 are approximately 1.1nM and 1.5nM, respectively. Finally, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs. The minimum detection limit of the assay was 6.2ng/ml CD44v3-10 and the linear range was up to 125ng/ml. Intra- and inter-assay coefficients of variation were 2.2% and 2.9%, respectively. The intra- and inter-assay recoveries were 99.3% and 105.3%, respectively. Taken together, these results suggest that this novel sandwich ELISA using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs will be useful for the detection of soluble CD44v3 in cancer patient sera.

Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal.

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8-10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation-related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

Expression analysis of CD44 isoforms S and V3, in patients with esophageal squamous cell carcinoma.

OBJECTIVES: CD44 is a member of the cell adhesion molecules family. Naturally, CD44S, along with CD44V3 influence the cell motility, migration, and adhesion, while in tumor cells they lead to tumor invasion, progression, and metastasis. The purpose of this research is to evaluate the CD44S and CD44V3 expression in Esophageal Squamous Cell Carcinoma (ESCC) and to reveal their correlations with clinicopathological features of patients. MATERIALS AND METHODS: Fresh tumoral and distant tumor-free esophageal tissues were obtained from 50 patients with ESCC. Using quantitative real-time PCR, the expression levels of CD44S and CD44V3 were quantified and compared in both groups of cells. The patients had not received any therapeutic interference, such as chemotherapy or radiation, prior to sampling. RESULTS: Significant overexpression of CD44S and CD44V3 mRNA was observed in 13 (26.0%, P=0.03) and 11 (22.0%, P=0.007) tumor specimens, respectively. The expression of the genes were significantly correlated not only with each other (P=0.0001), but also with differentiation grade of tumor (P=0.033), stage of tumor progression (P=0.003), and depth of tumor invasion (P=0.00). In addition, low level of CD44V3 mRNA expression was attended to be associated with tumor invasion. CONCLUSION: There is no correlation between CD44S expression with clinicopathological features of patients; however, simultaneous expression of these genes has an important effect on tumorigenesis.

MicroRNA-143 inhibits migration and invasion of human non-small-cell lung cancer and its relative mechanism.

BACKGROUND: MicroRNAs (miRNAs) play important roles in many biological processes, including cancer development. Among those miRNAs, miR-143 shows tumor-suppressive activity in some human cancers. However, the function and mechanism of miR-143 in lung cancer cells remains unknown. Here we explored the role of miR-143 in lung cancer. RESULTS: According to qRT-PCR, we found that miR-143 was notably down-regulated in 19 NSCLC tissues and 5 cell lines. In vitro experiments showed us that miR-143 could significantly suppress the migration and invasion of NSCLC cell lines while it had no effects on the growth of NSCLC cell lines, and in vivo metastasis assay showed the same results. Finally, we found that the mechanism of miR-143 inhibiting the migration and invasion of NSCLC might be through targeting CD44v3. CONCLUSIONS: The up-regulated miR-143 in lung cancer could significantly inhibit cell migration and invasion, and this might work through targeting CD44v3, which was newly identified by us.

Comparison of the Expression of Variants of CD44 between Node-positive and Node-negative Breast Carcinomas

BACKGROUND: The purpose of this study is to determine the value of CD44 and its splice variants as markers for the metastatic potential of infiltrating ductal carcinomas of the breast. METHODS: Tissue samples of infiltrating ductal carcinoma of the breast were examined for the expression of standard CD44 (CD44H) and s CD44 isoforms, v3, v4-5 and v6 in 41 node-positive and 31 node-negative cases. The immunohistochemistry results were correlated with other clinicopathologic parameters, and these results were correlated with accompanying high grade and non-high grade DCIS areas of the tumors in both node-positive and node-negative cases. RESULTS: The expression of CD44 in the invasive tumor areas and in the metastatic foci of the lymph nodes showed a statistically significant correlation. The expression of CD44H in the invasive tumor areas and the DCIS area showed a statistically significant correlation in the lymph node (-) group. There was statistical significance between the CD44 H and CD44v3 expressions and the histologic grade of the invasive tumor in the cases with positive lymph nodes. There was no statistical significance between CD44 expression and lymph node metastasis, tumor size and type of tumor margin. CONCLUSIONS: We conclude that changes in the CD44 expression in breast cancer occur early in breast carcinogenesis, and this is involved in tumor differentiation, but we could not establish any correlation between the expression of the CD44 variant isoforms and the metastasis of breast cancer.

Expressions of CD44v3 and CD54 in oral premalignant epithelia / 实用口腔医学杂志

Objective:To investigate the expressions of CD44v3 and CD54 in oral premalignant epithelia and its significance in the carcinogenesis of oral mucosa.Methods:The expressions of CD44v3 and CD54 in 19 cases of normal mucosa and mucosal hyperplasia,22 case of dysplastic epithelia,26 cases of oral cancer mucosa were detected by using immunohistochemistry.Results:CD44v3 and CD54 proteins showed no expression or micro-expression in normal and hyperplastic epithelia.The expressions increased in the dysplastic epithelia and mucosa of oral cancer gradually.The difference between the expressions of the three groups was significant(P

Inhibitory effects of shRNA targeting CD44v3 on invasion behavior of human colon cancer cell SW480 in vitro induced by hyaluronan / 中国癌症杂志

Purpose:To investigate the effect of short hairpin RNA (shRNA) targeting CD44v3 on adhesion and invasion behavior of human colon cancer cell line SW480 induced by hyaluronan in vitro. Methods:RNA interference plasmid including U6 promoter and expressing shRNA of CD44v3 was designed, constructed, and transfected into SW480 cell line. CD44v3 expression was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Adhesive and invasive ability were examined by plate adhesion model and Boyden chamber model.Results:After plasmid transfection,CD44v3 expression in SW480 cell and the number of SW480 cell induced by hyaluronan adhesiving on plate or permeating septum of Boyden chamber decreased significantly (P