Expression Of CD44 Variant Isoforms Research Articles

TRAF4/6 Is Needed for CD44 Cleavage and Migration via RAC1 Activation.

Simple SummaryTumor cells receive signals from the surrounding extracellular matrix that affect their growth and survival. An important component of the extracellular matrix is the large polysaccharide hyaluronan, which binds and activates certain receptors at the cell surface, including CD44. Activation of CD44 initiates several signaling pathways; one of them involves the cleavage of CD44 by proteases, leading to the release of the intracellular domain of CD44, which after translocation to the nucleus affects the transcription of certain genes. In the present report, we elucidate the mechanism by which CD44 is cleaved, and show that this occurs at an increased rate in stem-like tumor cells grown in spheres. We also show that CD44 cleavage promotes the migration of tumor cells. Since the mechanism we have elucidated promotes tumorigenesis, it is possible that inhibition of this pathway may be beneficial in the treatment of tumor patients.The hyaluronan receptor CD44 can undergo proteolytic cleavage in two steps, leading to the release of its intracellular domain; this domain is translocated to the nucleus, where it affects the transcription of target genes. We report that CD44 cleavage in A549 lung cancer cells and other cells is promoted by transforming growth factor-beta (TGFβ) in a manner that is dependent on ubiquitin ligase tumor necrosis factor receptor-associated factor 4 or 6 (TRAF4 or TRAF6, respectively). Stem-like A549 cells grown in spheres displayed increased TRAF4-dependent expression of CD44 variant isoforms, CD44 cleavage, and hyaluronan synthesis. Mechanistically, TRAF4 activated the small GTPase RAC1. CD44-dependent migration of A549 cells was inhibited by siRNA-mediated knockdown of TRAF4, which was rescued by the transfection of a constitutively active RAC1 mutant. Our findings support the notion that TRAF4/6 mediates pro-tumorigenic effects of CD44, and suggests that inhibitors of CD44 signaling via TRAF4/6 and RAC1 may be beneficial in the treatment of tumor patients.

Immunohistochemical expression of CD44v9 and 8-OHdG in ovarian endometrioma and the benign endometriotic lesions adjacent to clear cell carcinoma.

Expression of CD44 variant isoforms (CD44v) promotes the synthesis of reduced glutathione and contributes to reactive oxygen species defense through up-regulation of the intracellular antioxidant. The aim of the study was to investigate the expression of CD44v9 and oxidative DNA damage marker, 8-OHdG, in benign ovarian endometrioma (OE) and OE harboring clear cell carcinomas (CCC). A retrospective study was performed at the Department of Gynecology, Nara Medical University hospital from January 2006 to December 2012. Patients with histologically confirmed benign OE (n = 27) and OE harboring areas of CCC (n = 8) were selected. Tissue samples were immunohistochemically analyzed for the presence of CD44v9 and 8-OHdG using avidin-biotin complex method. CD44v9 was located on the cell membrane of endometriotic epithelial cells and expressed in 88.9% (24/27) of benign OE tissues. Only 25.0% (2/8) of benign endometriotic lesions adjacent to CCC was found to stain weakly for CD44v9. Percentage of CD44v9 positive cells was 68.5 ± 20.2% (mean ± standard deviation) of benign OE, 16.7 ± 16.5% of CCC endometriotic tissue (P < 0.001). Compared to benign OE, CCC endometriotic tissue showed a significant increase in the proportion of 8-OHdG expression (77.3 ± 22.5% vs 94.9 ± 3.0%, P = 0.049). A significant negative correlation was observed between CD44v9 status and 8-OHdG nuclear expression (r = -0.458, P = 0.006). Alterations in CD44v9 and 8-OHdG may be associated with malignant transformation of benign OE.

Prognostic value of CD44 variant isoform expression in dogs with multicentric high-grade B-cell lymphoma.

OBJECTIVE To determine the prognostic value of CD44 variant isoform expression in dogs with multicentric high-grade B-cell lymphoma (BCL). ANIMALS 45 dogs with multicentric BCL and 10 healthy control Beagles. PROCEDURES The medical record database of a veterinary teaching hospital was searched to identify dogs with BCL that were treated between November 2005 and April 2015. Information regarding overall response to chemotherapy, progression-free survival (PFS) time, and overall survival time was extracted from each record. Archived lymph node aspirate specimens from dogs with BCL and lymph node aspirate specimens from the 10 control dogs underwent real-time PCR analysis to determine mRNA expression of CD44 variant isoforms of exons 3, 6, and 7 and the CD44 whole isoform. For each isoform, mRNA expression was compared between dogs with BCL and control dogs. The mean relative expression of each isoform was used to classify dogs with BCL into either a high- or low-expression group, and overall response rate, PFS time, and overall survival time (ie, indices of prognosis) were compared between the 2 groups. RESULTS For all isoforms evaluated, mean relative mRNA expression for dogs with BCL was numerically lower than that for control dogs. Dogs with BCL and high CD44 isoform expression had a lower overall response rate, median PFS time, and median overall survival time, compared with dogs with BCL and low CD44 isoform expression. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, for dogs with BCL, high expression of exons 3, 6, and 7 was associated with a poor prognosis.

CD44 variant–dependent regulation of redox balance in EGFR mutation–positive non–small cell lung cancer: A target for treatment

ObjectivesThe regulation of redox balance in cancer cells is an important factor in tumor development and chemoresistance, with oncogene activation having been shown to induce the generation of reactive oxygen species (ROS). Activating mutations of the epidermal growth factor receptor gene (EGFR) are oncogenic drivers in non–small cell lung cancer (NSCLC), but it has remained unknown whether ligand-independent EGFR signaling conferred by EGFR mutation triggers ROS generation in NSCLC cells. Materials and MethodsHEK293T cells were transfected with an expression vector for mutant EGFR. The expression of CD44 variant (CD44v) isoforms in NSCLC cell lines was evaluated by flow cytometry. Cells were depleted of CD44v by RNA interference and assayed for ROS and glutathione (GSH) levels. The effect of CD44v on cisplatin sensitivity was evaluated in vitro with the MTS assay. ResultsEGFR signaling due to EGFR mutation increased ROS levels in transfected HEK293T cells. The expression of CD44v isoforms was found to be inversely correlated with basal ROS levels in EGFR mutation–positive NSCLC cell lines. Knockdown of CD44v induced depletion of intracellular GSH and increased ROS levels in EGFR-mutated NSCLC cells that express CD44v at a high level (CD44vhigh). In addition, depletion of GSH by treatment with buthionine-[S, R]-sulfoximine induced marked accumulation of ROS and enhanced the cytotoxicity of cisplatin in CD44vhighEGFR-mutated NSCLC cells but not in corresponding CD44vlow cells. This enhancement of cisplatin cytotoxicity by GSH depletion was prevented by treatment with the antioxidant N-acetyl-l-cysteine. Knockdown of CD44v also enhanced cisplatin cytotoxicity in CD44vhighEGFR mutation–positive NSCLC cells but not in CD44vlow cells. ConclusionOur results thus implicate CD44v in redox adaptation and as a potential target for treatment in CD44vhighEGFR-mutated NSCLC cells.

Abstract 857: ELF3 modulates mesenchymal to epithelial transition through regulation of CD44 variant isoform expression in pancreatic cancer cells

Abstract BACKGROUND: Epithelial to Mesenchymal Transition (EMT) and Mesenchymal to Epithelial Transition (MET) may participate in pancreatic ductal adenocarcinoma (PDAC) metastasis. CD44 proteins are cell membrane proteins which function as co-receptors regulating many cellular functions. Some reports have shown that isoform switching from CD44 variant form (CD44v) to CD44 standard form (CD44s) takes place during the EMT. (Brown et al., J. Clin. Invest, 2011, Yae et al., Nat Commun., 2012). The AIM of this study was to explore the regulation of CD44 isoform switching in PDAC cells. Methods: The expression levels of CD44v and CD44s in PDAC cell lines were assessed by immunohistochemistry and immunoblot analysis. PDAC cell lines were implanted into immunodeficiency mice and treated with sulfasarazine for 14 days. Sulfasarazine, which inhibits glutamate-cysteine transport, reduces CD44v-dependent cell growth. CD44v or CD44s positive cells were isolated using flow cytometry and induced to undergo the EMT by TGF-β treatment (7.5ng/mL) for 72 hours. A doxycycline inducible system was employed to overexpress Slug, which promotes the EMT phenotype. To induce MET, the cells were released from TGF-β treatment and cultured for an additional 72 hours. E74-like ETS Transcription Factor 3 (ELF3) expression was knocked-down by siRNA transfection. RESULTS: Immunohistochemistry analysis revealed that CD44v protein is expressed specifically by PDAC with ductal structures within the complex tumor environment of these cancers. However, in contrast, CD44v expression was not observed in solitary infiltrating cancer cells. PANC-1, AsPC-1 and SUIT-2 PDAC cell lines expressed both CD44v and CD44s isoforms. Tumor mass in implanted PANC-1, AsPC-1 and SUIT-2 cells were reduced 69%, 50% and 36% respectively by sulfasarazine treatment following 2 weeks of therapy. However, CD44v null HPAC cells were resistant to sulfasarazine therapy following implantation. E-cadherin was expressed in CD44v positive cells but not in CD44s positive cells, and both CD44v and E-cadherin protein were reduced by TGF-β treatment. Slug over expressed cells were resistant to sulfasarazine treatment compared to non-Slug-induced cells. Removal of TGF-β restored both CD44v and E-cadherin expression levels which consistently occurs with MET. However, restoration of CD44v expression was diminished when ELF3 expression was reduced by siRNA transfection. CONCLUSION: The isoform switching between CD44v and CD44s was reversible. The expression of CD44v isoform appears to be controlled by ELF3 during MET. Citation Format: Ayano Kabashima-Niibe, Hiromasa Takaishi, Takanori Kanai, Hideyuki Saya, Gregory J. Gores, Hajime Higuchi. ELF3 modulates mesenchymal to epithelial transition through regulation of CD44 variant isoform expression in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 857. doi:10.1158/1538-7445.AM2017-857

Expression of CD44 variant isoforms, CD44v3 and CD44v6, are associated with prognosis in nasopharyngeal carcinoma.

The clinical and prognostic significance of CD44 variant isoform expression in nasopharyngeal carcinoma is not well known. This study aimed to clarify whether CD44 variant isoform expression serves as a prognostic factor in nasopharyngeal carcinoma. Forty-two nasopharyngeal carcinoma patients, who underwent concurrent chemoradiotherapy as the initial treatment, were the subjects of investigation. Expression of CD44 variant isoforms, CD44v3, CD44v4, CD44v5, CD44v6 and CD44v7, in nasopharyngeal carcinoma was assessed in relation to concurrent chemoradiotherapy resistance and disease-specific survival of the patients. The patients with CD44v6 high expression showed a clinically incomplete response to concurrent chemoradiotherapy at the primary site. The disease-specific survival rate was lower in patients with high expression of CD44v3 than in those with low expression. These results suggest that analysis of CD44v6 and CD44v3 expression is useful in estimating prognosis and determining effective treatment strategies in nasopharyngeal carcinoma.

CD44, Hyaluronan, the Hematopoietic Stem Cell, and Leukemia-Initiating Cells.

CD44 is an adhesion molecule that varies in size due to glycosylation and insertion of so-called variant exon products. The CD44 standard isoform (CD44s) is highly expressed in many cells and most abundantly in cells of the hematopoietic system, whereas expression of CD44 variant isoforms (CD44v) is more restricted. CD44s and CD44v are known as stem cell markers, first described for hematopoietic stem cells and later on confirmed for cancer- and leukemia-initiating cells. Importantly, both abundantly expressed CD44s as well as CD44v actively contribute to the maintenance of stem cell features, like generating and embedding in a niche, homing into the niche, maintenance of quiescence, and relative apoptosis resistance. This is surprising, as CD44 is not a master stem cell gene. I here will discuss that the functional contribution of CD44 relies on its particular communication skills with neighboring molecules, adjacent cells and, last not least, the surrounding matrix. In fact, it is the interaction of the hyaluronan receptor CD44 with its prime ligand, which strongly assists stem cells to fulfill their special and demanding tasks. Recent fundamental progress in support of this “old” hypothesis, which may soon pave the way for most promising new therapeutics, is presented for both hematopoietic stem cell and leukemia-initiating cell. The contribution of CD44 to the generation of a stem cell niche, to homing of stem cells in their niche, to stem cell quiescence and apoptosis resistance will be in focus.

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

Osteopontin-mediated enhanced hyaluronan binding induces multidrug resistance in mesothelioma cells

Malignant pleural mesothelioma (MPM) is resistant to chemotherapy and thus shows a dismal prognosis. Osteopontin (OPN), a secreted noncollagenous and phosphoprotein, is suggested to be involved in the pathogenesis of MPM. However, the precise role of OPN, especially in the multidrug resistance of MPM, remains to be elucidated. We therefore established stable transfectants (ACC-MESO-1/OPN), which constitutively express OPN, to determine its role in the chemoresistance observed in MPM. The introduction of the OPN gene provides MPM cells with upregulated multidrug resistance through the mechanism of enhanced hyaluronate (HA) binding. The expression of CD44 variant isoforms, which inhibit HA binding, significantly decreased in ACC-MESO-1/OPN cells in comparison to control transfectants. Interestingly, the inhibition of the HA-CD44 interaction abrogated multidrug resistance in the ACC-MESO-1/OPN, thus suggesting the involvement of the surviving signal emanating from the HA-CD44 interaction. An enhanced level of the p-Akt in ACC-MESO-1/OPN cells was observed, and was diminished by CD44 siRNA. Inhibition of the Akt phosphorylation increased in number of the cells underwent apoptosis induced by NVB, VP-16 and GEM. Collectively, these results indicate that OPN is strongly involved in multidrug resistance by enhancing the CD44 binding to HA.

CD44 variant isoforms in head and neck squamous cell carcinoma progression

The CD44 family of receptors includes multiple variant isoforms, several of which have been linked to malignant properties including migration, invasion, and metastasis. The objective of this study was to investigate the role of the CD44 v3, v6, and v10 variant isoforms in head and neck squamous cell carcinoma (HNSCC) tumor progression behaviors. Laboratory study involving cell cultures and clinical tissue specimens. Analysis of the expression of standard CD44s and the CD44 variant isoforms v3, v6, and v10 was carried out in the HNSCC cell line, HSC-3. The role of CD44 isoforms in migration, proliferation, and cisplatin resistance was determined. Immunohistochemical analysis was performed on clinical tissue specimens obtained from a series of 82 HNSCC patients. The expression of standard CD44s and the CD44 v3, v6, and v10 variants in primary tumor specimens (n = 82) and metastatic cervical lymph nodes (n = 24) were analyzed with respect to various clinicopathologic variables. HSC-3 cells express at least four CD44 isoforms, and these CD44 isoforms mediate migration, proliferation, and cisplatin sensitivity. Compared with primary tumors, a greater proportion of metastatic lymph nodes demonstrated strong expression of CD44 v3 (lymph node 14/24 vs. primary tumor 38/82), CD44 v6 (lymph node 18/24 vs. primary tumor 26/82), and CD44 v10 (lymph node 14/24 vs. primary tumor 16/82), while expression of standard CD44 was not significantly different in metastatic lymph nodes and primary tumors (lymph node 10/24 vs. primary tumor 60/82). Expression of CD44 variant isoforms were associated with advanced T stage (v3 and v6), regional (v3) and distant (v10) metastasis, perineural invasion (v6), and radiation failure (v10). CD44 v6 and CD44 v10 were also significantly associated with shorter disease-free survival. CD44 isoforms mediate migration, proliferation, and cisplatin sensitivity in HNSCC. Furthermore, expression of certain CD44 variants may be important molecular markers for HNSCC progression and should be investigated as potential therapeutic targets for therapy.

The involvement of osteopontin and its receptors in multiple myeloma cell survival, migration and invasion in the murine 5T33MM model

Multiple myeloma (MM) is a malignancy characterised by the accumulation of monoclonal plasma cells in the bone marrow. Different reports indicate the expression of CD44 variant isoforms (CD44v) by MM cells. Osteopontin (OPN), which is expressed by MM cells, is known to be a ligand for CD44v6. In this study, we investigated the role of OPN with emphasis on a functional correlation between OPN and CD44v in the 5T33MM model. Our group reported the expression of CD44v by 5T33MM cells. Using this model, we have demonstrated the secretion of OPN by 5T33MM cells. OPN affected 5T33MM cell survival by increasing proliferation and inhibiting apoptosis. OPN also stimulated 5T33MM cell migration, transendothelial migration and matrix metalloproteinase-9 activity. We confirmed the proliferative and migratory effects of OPN on human MM cells. By applying inhibiting anti-CD44v6 antibodies, we found that OPN stimulated cell proliferation by engaging this isoform. Anti-CD44v antibodies and RGD peptides both inhibited cell migration, suggesting an involvement of both, CD44v isoforms and integrins. In conclusion, OPN may act as a mediator of MM cell survival by engaging CD44v. The protein is further involved in migration and invasion of MM cells through the activation of either alphavbeta3 integrin or CD44v isoforms.

Short-term treatment with anti-CD44v7 antibody, but not CD44v4, restores the gut mucosa in established chronic dextran sulphate sodium (DSS)-induced colitis in mice

Increased expression of CD44 variant isoforms have been shown on the inflammatory infiltrates in human and mouse colitis and blockade or deletion of CD44 isoforms inhibit experimental colitis. The objective of this study was to find out if short-term treatment of CD44 antibodies specific to CD44v7, but not to other variant isoforms, suppresses leucocyte-endothelial interaction in chronic dextran sodium sulphate (DSS)-induced colitis in mice. Chronic colitis was induced by oral administration of four cycles of 5% DSS in BALB/c mice. Expression of CD44 was investigated on isolated mononuclear cells of the gut immune system. In established colitis, mice were treated with antibodies against CD44v7 or CD44v4 three times in 7 days. Intravital microscopy was used to study leucocyte-endothelial interactions and leucocyte extravasation. As a marker of inflammatory infiltrates myeloperoxidase was quantified in gut tissue. CD44-induced apoptosis was determined by fluorescence staining of hypodiploidic cell nuclei. In chronic DSS-induced colitis both CD44 variant isoforms, v4 and v7 were significantly up-regulated on mononuclear cells. However, whereas anti-CD44v7 antibody treatment induced a marked restoration of the gut mucosa and significantly reduced endothelial sticking and extravasation of circulating leucocyte in vivo (P < 0.01), application of anti-CD44v4 or an isotype control antibody had no anti-inflammatory effect. A significant reduction of myeloperoxidase activity was detected after blockade of CD44v7, but not v4. Short-term treatment with anti-CD44v7 antibody blocks T cell extravasation and recruitment to the intestinal mucosa and cures established experimental colitis.

Simultaneous bilateral breast carcinoma: Histopathological characteristics and CD44/catenin-cadherin profile.

Family history of breast carcinoma, multicentric tumor foci in one breast, and in situ lobular carcinoma increase the risk of bilateral breast cancer (BBC), synchronous or metachronous. Synchronous tumors are designated as simultaneous breast carcinoma if they appear at the same time. The CD44 family and cadherin/catenin immunophenotype of this group of BBCs has not yet been evaluated. The aim of this study was to compare clinicopathological characteristics and immunohistochemical profiles of simultaneous BBC and corresponding lymph node metastases in eight patients. In toto 15 primary and 9 metastatic tumors were evaluated. The expression of CD44 variant isoforms, beta-catenin, E, P and N-cadherin were evaluated by immunohistochemistry. Rare types of breast carcinoma were frequent in this group of patients. There were 6 pleomorphic lobular, 5 invasive ductal of usual type, 3 atypical medullary carcinomas, 2 mucinous and one invasive micropapillary carcinoma. The expression CD44v6 was most frequent, followed by CD44v3-10, CD44v5, and CD44v3. CD44v4 was generally not expressed. E-cadherin was expressed in 80% primary tumors, 40% expressed N-cadherin, and 66% expressed P-cadherin. Generally, simultaneous carcinomas had different morphology and different immunophenotype. Each primary tumor was more similar to its corresponding metastatic tumor than to the contralateral primary tumor.

Prognostic significance of CD44 expression in diffuse large B cell lymphoma of activated and germinal centre B cell-like types: a tissue microarray analysis of 90 cases

Background: Gene expression profiling of diffuse large B cell lymphoma (DLBCL) revealed three disease types: germinal centre B cell-like (GC), activated B cell-like (ABC), and a “third” type. Expression of...

Transient CD44 Variant Isoform Expression and Reduction in CD4+/CD25+ Regulatory T Cells in C3H/HeJ Mice with Alopecia Areata

Alopecia areata, an autoimmune disease affecting anagen stage hair follicles, can be induced by grafting spontaneous alopecia areata affected skin to normal-haired C3H/HeJ mice. As the onset of alopecia areata can be significantly retarded by anti-CD44 variant isoform 10 treatment, it was interesting to explore the underlying disease mechanism. Two weeks after transplanting alopecia areata affected skin, expression of CD44 variant isoforms 3, 6, 7, and 10 was strikingly upregulated as compared with sham-grafted mice. By 6 wk after grafting, CD44 variant isoform levels had returned to normal, whereas in draining lymph nodes, CD44 variant isoform expression was slightly decreased. Leukocytes in the skin of mice with chronic alopecia areata expressed a hematopoietic isoform of CD44 and CD44 variant isoform 6 at an elevated level, but CD44 variant isoform 3 expression was reduced. Cytokine expression in leukocytes of chronic alopecia areata affected skin was higher than in normal-haired controls. Cytokine expression also increased postsurgery in sham and alopecia areata grafted mice, but remained elevated only in mice receiving alopecia areata affected skin. Finally, from the skin of mice with chronic alopecia areata and of mice transplanted with alopecia areata affected skin, an increased number of CD4(+) and CD8(+) cells, but a strongly decreased number of CD4(+)/CD25(+) regulatory T cells was recovered. Thus, expression of CD44 variant isoforms is important for the migration of leukocytes during the initial period of alopecia areata. CD44, however, is apparently not involved in the maintenance of the disease state, which is characterized by high cytokine expression levels, an increased number of CD4(+) and CD8+ cells, but a low level of CD4(+)/CD25(+) suppressor cells.

Expression of CD44 variant isoforms and association to the benign form of endocrine pancreatic tumours

The expression of CD44 and its isoforms have been shown in many neoplastic tissues to serve as prognostic indicators, therefore, the feasibility of using these as prognostic markers in endocrine pancreatic tumour patients was examined. Immunohistochemistry (IHC) was performed on 26 tumour samples (5 gastrinomas, 3 glucagonomas, 10 non-functioning tumours, 6 insulinomas, 2 mixed insulinoma and glucagonomas) with monoclonal antibodies against CD44s (standard form) and variant isoforms (v4, v5, v6, v7, v7-8, v9, v10). Staining was correlated to the tumour proliferation, malignancy, metastasis and patients survival. There was variable expression of CD44s. All tumours showed complex expression of many isoforms. CD44v6 and CD44v9 were down regulated in malignant tumours. There was statistical significance of CD44v6 expression in benign tumours (P < 0.05) compared to malignant tumours and near significance in CD44v9 expression (P = 0.0574). Survival of the patients with CD44v6 positive staining was higher than those who were negative (P = 0.0822). Moreover, the expression was well correlated to the patients without any distant metastases (CD44v6, p < 0.001; CD44v9, P < 0.01). Tumour proliferation (Ki67 index) correlated directly to the malignancy (P < 0.05) and there was inverse correlation between Ki67 index and CD44v6 (P < 0.05) as well as v9 (P < 0.05). Endocrine pancreatic tumours express CD44s and isoforms differentially. Expression of the two isoforms of CD44, namely v6 and v9 seem to be related more to benign form of the tumour and could serve as a predictor of good prognosis.

Expression of CD44 variant isoforms in bronchial precancerous lesions.

To explore the role of CD44 variant isoforms (CD44v) expression on oncogenesis of squamous cell carcinoma of the lung. CD44v expression was detected by immunohistochemical analysis in cancer tissues and precancerous lesion samples of 34 patients with squamous cell carcinoma of the lung and its expression time and frequency were studied during carcinogenesis of lung cancer. Membrane CD44v were present in 0 of 12 normal mucosae adjacent to the cancers , 2 of 14 squamous metaplasias , 3 of 13 mild dysplasias , 5 of 17 moderate dysplasias , 12 of 21 severe dysplasias , 5 of 9 carcinomas in situ , and 21 of 34 invasive tumors. With the progress of bronchial lesions from mild dysplasia to severe dysplasia and to carcinoma in situ and invasive carcinoma , the positive rate of CD44v expression increased gradually. The research indicates that CD44v expression is likely to be an usable marker to judge the probability of squamous cell carcinoma of the lung and that routine assessment of CD44v expression may be valuable for the early diagnosis of squamous cell carcinoma of the lung.

Predominant Expression of CD44 Splice Variant v10 in Malignant and Reactive Human Skin Lymphocytes

The remarkable functional diversity of the cell surface receptor CD44 may be due to expression of multiple variant isoforms generated by alternative splicing of variant exons. Functional and correlative data implicate a role of CD44 variant isoforms in adhesion dependent processes such as lymphocyte recirculation and tumor progression and metastasis. We have analyzed 25 primary cutaneous lymphomas and 35 reactive lymphoid cell skin infiltrates or T cell-mediated skin diseases for the expression of CD44 variant isoforms. Irrespective of histologic typing, staging, and grading, cutaneous lymphomas as well as nonmalignant skin-infiltrating CD3+ CD4+ and CD8+ T and CD19+ B lymphocytes exhibited a strong expression of CD44v10 and a moderate expression of CD44v3 as determined by immunohistochemistry, immunofluorescence microscopy, and mRNA analysis. Expression of v5, v6, v7, and v9-containing CD44 variant isoforms was not detected. Furthermore, flow cytometry revealed expression of CD44v10 on a significant proportion of peripheral blood lymphocytes from Sézary's syndrome patients and a remarkable co-expression with cutaneous lymphocyte antigen. These results indicate a distinct CD44 variant isoform expression pattern associated with skin-homing lymphocytes different to lymphatic cells at noncutaneous sites. This differential expression pattern of CD44 variant isoforms may contribute to the development of lymphocyte skin infiltrates and/or the unique biologic behavior of cutaneous lymphomas.

Correlation of CD44 expression with proliferative activity of normal human breast epithelial cells in culture.

A number of studies have shown that certain variant isoforms of CD44 are overexpressed in human breast cancer, suggesting their use as indicators of the presence of malignant cells. We now show that CD44 isoform mRNA and protein expression is upregulated in normal human breast epithelial cells (HBEC) when these cells are stimulated to proliferate in culture. Reverse transcription-PCR analysis of cultured normal HBEC revealed complex patterns of CD44 mRNA expression that were indistinguishable from patterns previously shown to be characteristic of tissue samples containing malignant HBEC. CD44v6-expressing cells were identified in cultures generated from FACS-purified populations of either normal luminal (CALLA-MUC-1+) or myoepithelial (CALLA+MUC-1-) cells, even though immunohistochemical analysis of normal breast tissue sections confirmed CD44v6 expression to be limited to the myoepithelium in vivo. Increased expression of both CD44v mRNA and protein in cultured populations of normal HBEC was shown to correlate positively with the proportion of cells that were proliferating (Ki-67+) independent of cell density. These results indicate that activation of CD44 variant isoform expression in HBEC occurs as a normal response to factors that stimulate their proliferation and suggests caution in the use of this marker to identify malignant cells.

Expression of CD44 variant isoforms in leukocytes of the joint fluid of rheumatoid arthritis patients and the effect of steroids.

Several CD44 variant isoforms have been reported in the synovium of patients with rheumatoid arthritis (RA), but the physiological and pathological roles of these isoforms have not been identified. In this study, we investigated the expression of CD44 variant isoform mRNA using the reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan PCR methods in leukocytes of blood and joint fluid obtained from the knee joints of 20 RA patients. We also examined the effects of steroids (prednisolone and/or dexamethasone palmitate) and some disease-modifying antirheumatic drugs (DMARDs) on the expressions of CD44 variants. The expressions of CD44H mRNA and CD44V10 mRNA of leukocytes were significantly higher in both the blood and joint fluid samples of the RA patients compared with the samples of healthy volunteers. The level of CD44V10 mRNA in the leukocytes of the joint fluid was higher than that in the blood of the RA patients. These results suggest that the increase of CD44V10 mRNA expression in the leukocytes of RA patients can induce a focusing of leukocytes on synovial tissue and an infiltration of leukocytes into the joint fluid. In the steroid-treated RA patients, the expression of CD44H mRNA in the leukocytes was significantly decreased in the joint fluid samples, whereas the expression of CD44V10 mRNA of leukocytes was a higher level. None of the DMARDs used here showed any influence on the expressions of CD44 variants. These results suggest that steroid treatment affects CD44H mRNA expression, whereas steroids and DMARDs have no influence on CD44V10 mRNA expression. Therefore, only the suppression of CD44H mRNA expression will not be enough to control RA. It is possible that the expression of CD44V10 is associated with a pathway of RA immunological processes.