Regulation Of CD44 Expression Research Articles

Effects of Siraitia grosvenorii extract on nonalcoholic steatohepatitis-like lesions in Sprague Dawley rats fed a choline-deficient, methionine-lowered, l-amino acid-defined diet.

Siraitia grosvenorii is the fruit of a cucurbitaceous vine endemic to China. Its extract has been used as a sweetener and exhibits various anti-inflammatory and anticarcinogenic effects mediated via its antioxidant properties. In the present study, we aimed to clarify the preventive or ameliorative effects of S. grosvenorii extract (SGE) on nonalcoholic steatohepatitis-like lesions induced in male Hsd: Sprague Dawley rats fed a choline-deficient, methionine-lowered, l-amino acid-defined diet for 13 weeks. This diet increased hepatotoxicity parameters and upregulated the expression of inflammation- and fibrosis-related genes in the liver, resulting in the progression of hepatic lesions, oxidative stress, hepatocellular apoptosis, and fibrosis. Furthermore, this diet upregulated the expression of phosphorylated nuclear factor-κB (NF-κB) and CD44. SGE administration inhibited these lesions, similar to CD44, a factor that controls hepatic inflammation and fibrosis. These results revealed that SGE impacts the disease stage via antioxidative effects and regulation of CD44 expression. SGE was found to be useful for preventing and treating steatohepatitis.

HPV DOWN-REGULATES THE STEM CELL MARKER CD44 IN VIRAL-RELATED ORAL EPITHELIAL DYSPLASIA AND HNSCC

Objective Head and Neck Squamous Cell Carcinoma (HNSCC) represents the sixth most common malignancy worldwide and is characterized by dismal prognosis and poor patient survival. More than 75% of HNSCCs arise on a precancerous lesion. CD44 is a membrane bound glycoprotein stem-cell marker strongly expressed in normal oral mucosal epithelium. Upregulated in HNSCC, CD44 participates in key cell functions including cell division, migration and adhesion, and is recognized as a negative prognosticator for the disease. In addition, HPV(+) tumors show decreased CD44 levels when compared to HPV(-) neoplasms. We aimed to investigate the role of HPV infection in the regulation of CD44 expression in oral epithelial dysplasia (OED) and invasive HNSCC. Methods Formalin fixed, paraffin embedded specimens of HPV(+) OED (N=16), HPV(-) OED (N=15) and HNSCC (N=29) were evaluated by immunohistochemistry for CD44. Among the carcinoma specimens, five were HPV(+) and 24 HPV(-); 13 well-differentiated (WD), 5 moderately-differentiated (MD) and 6 poorly-differentiated (PD). HPV positivity was confirmed by immunohistochemistry for the surrogate marker p16. CD44 immunoreactivity was semi-quantitatively evaluated. Statistical analysis was performed using one-way ANOVA. Results HPV(+) OEDs (mean expression=1.74) showed significantly lower CD44 membranous immunoexpression than HPV(-) OEDs (mean expression=2.42, p Conclusions Lower CD44 expression in HPV(+) OEDs and HNSCCs may reflect decreased numbers of stem cells in HPV-driven lesions. The latter, can explain the limited frequency of malignant transformation in HPV(+) OEDs and better survival rates for patients with HPV(+) tumors.

The Vascular Bone Marrow Niche Influences Outcome in Chronic Myeloid Leukemia

The endosteal bone marrow niche is known to protect leukemic stem cells (LSC) from chemotherapy, but the role of the vascular niche in the bone marrow microenvironment in leukemia is largely unknown. E-selectin, which is expressed on bone marrow endothelial cells, has been described to regulate the dormancy of normal hematopoietic stem cells (HSC) by increasing HSC proliferation (Winkler IG, 2012). In chronic myeloid leukemia (CML), one of the myeloproliferative neoplasias caused by the oncogene BCR-ABL1, E-selectin (Krause DS, 2014) and the adhesion molecule CD44, which is overexpressed on CML-initiating cells (Krause DS, 2006) and to which E-selectin binds (Videira PA, 2018), was shown to be essential for homing and engraftment of LSC. However, exact mechanisms of the interactions between BCR-ABL1, CD44 expression and interactions with the vascular niche are unclear.Hypothesizing that E-selectin influences the cell cycle of leukemic stem cells (LSC) in CML leading to improved eradication of LSC in case of treatment with the tyrosine kinase inhibitor imatinib, we treated murine recipients of CML-initiating cells in the retroviral transduction/transplantation model with the E -selectin inhibitor GMI-1271 (uproleselan, GlycoMimetics, Inc.) and imatinib, considered standard of care in CML. This increased the survival of mice compared to animals treated with imatinib alone and decreased the engraftment of CML-initiating cells in this model, as well as in the majority of mice in a xenotransplantation model of NOD SCID IL2 receptor gamma knockout (NSG) mice injected with human CML cells and treated with uproleselan. Treatment of NSG mice with uproleselan also decreased the contact time of injected human CML cells with the bone marrow endothelium. As shown in in vitro and in vivo experiments treatment with GMI-1271 and non-adhesion to E-selectin increased the cell cycle of BCR-ABL1+ LSC with a concomitant increase in expression of the transcriptional regulator and protooncogene Scl/Tal1. SCL/TAL1 was shown to negatively regulate CD44 expression by binding to the CD44 regulatory element, which was increased in presence of imatinib. In addition, we demonstrated SCL/TAL1 to be an indirect phosphorylation target of BCR-ABL1 and a negative transcriptional regulator of CD44 expression.In confirmation of our findings in mice, we also showed a negative correlation between SCL/TAL1 and CD44 expression in leukocytes of human patients with CML and that increased SCL/TAL1 expression is associated with decreased probability of relapse and increased relapse-free survival in human CML patients after allogeneic hematopoietic stem cell transplantation.In summary, inhibition of E-selectin in murine CML may lead to ‘non-adherence’ of LSC to the vascular niche and improved eradication by imatinib. This is likely due to an increase in cell cycle and an increase of Scl/Tal1 expression, which is shown to be regulated by BCR-ABL1. These data connect the adhesion of LSC in CML to the vascular niche via CD44 with the regulation of CD44 expression by SCL/TAL1 and BCR-ABL1, offering a potential new avenue for treatment. DisclosuresKrause:Glycotope: Consultancy; Glycomimetics Inc. until 06/2016: Consultancy, Research Funding; Merck KgGA in future: Research Funding; European Patent: Patents & Royalties: European Patent No. 16187926.7-1401 “FIBRONECTIN FOR USE IN THE TREATMENT OF LEUKEMIA” .

Regulation of CD44 Expression by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

Regulation of CD44 Expression by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

Activated ERK1/2 increases CD44 in glomerular parietal epithelial cells leading to matrix expansion

The glycoprotein CD44 is barely detected in normal mouse and human glomeruli, but is increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an invivo and invitro approach. Experimental FSGS was induced in CD44 knockout and wild-type mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain), and collagen IV staining were lower in CD44 knockout compared with wild-type mice with FSGS. Parietal epithelial cells had lower migration from Bowman's capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cell migration. Because our results showed de novo co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two invitro strategies were employed to prove that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44-overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is a regulator of CD44 expression, and increased CD44 expression leads to a pro-sclerotic and migratory parietal epithelial cell phenotype.

The regulation of CD44 expression by new tumor suppressor gene Arid2 and the influence of Arid2 on the invasion and metastasis in hepatocellular carcinoma cells

To investigate the effect of a new tumor-suppressor gene Arid2 on the expression level of CD44 and the mechanism that Arid2 regulates the invasion and metastasis in human hepatocellular carcinoma cells HepG2 and Huh7. Recombinant pGL3-CD44 reporter plasmids were transfected into hepatoma cell lines HepG2 and Huh7 cells infected with adenovirus Arid2(Ad-Arid2). Dual luciferase assays were used to determine the relative luciferase activities of reporter plasmids. Western blot technique was used to detect the influence of Arid2 on the expression of transmembrane glycoprotein CD44. Cell migration assays of tumor cells were employed to observe the impact of overexpression of Arid2 on the invasion and metastasis abilities of tumor cells. The sizes of transplanted tumors were recorded to observe the growth of subcutaneous transplanted tumors in nude mice. Statistical significance was analyzed by one-way ANOVA for multiple comparisons, and independent-samples t-test was utilized to compare two groups. Luciferase assay showed cells were transfected with different length of CD44 reporter plasmids, and their relative luciferase activities were improved to different degrees, compared with pGL3-Basic control. Meanwhile, the mean luciferase activities of pGL3-CD44 -791~+224bp reporter plasmids were significantly repressed by the overexpression of Arid2 which inhibition rates were up to 73.83%±0.92%(P< 0.05, HepG2) or 48.99%±1.37% (P <0.05, Huh7), compared with Ad-GFP control. Western blot results showed that CD44 protein expression was obviously decreased by overexpression of Arid2. Cell migration assays confirmed that the invasion and metastasis abilities were inhibited by increasing Arid2 expression in Human HepG2 or Huh7 cells, which inhibition rates were 66.95%±0.59%(P< 0.05)in HepG2 cells and 73.86%±0.49%(P< 0.05) in Huh7 cells respectively. The animal experiment results indicated that Arid2 could obviously delay or restrict the subcutaneous transplanted tumors growth in nude mice, which was declined by 98.57%±0.34%(P< 0.05). CD44 promoter activities and protein expressions were significantly down-regulated by Arid2 in vitro. The growth and metastasis of tumors were obviously restrained in the hepatocellular carcinoma cells and nude models. In brief, these researches indicate CD44 may play important roles in the process where the invasion and metastasis of hepatocellular carcinoma cells are under the control of Arid2.The studies introduce and evaluate the relationships between Arid2 and CD44, and further provide a new research direction with the occurrence and development of hepatocellular carcinoma.

Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells.

The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies.

Low-dose interleukin-8 induces the adhesion, migration and invasion of the gastric cancer SGC-7901 cell line.

Interleukin-8 (IL-8), an important inflammatory cytokine, is strongly associated with gastric cancer development and metastasis. High-dose (>1 ng/ml) IL-8 has been revealed to promote the adhesion, migration and invasion of human gastric cancer SGC-7901 cells in a dose-dependent manner. However, the IL-8 level produced by gastric cells is marginal, at even less than 1 ng/ml. It is unclear whether low-dose IL-8 also induces these capacities. In the present study, the effect of low-dose IL-8 on the adhesion, migration and invasion of the SGC-7901 cell line and the underlying molecular mechanism with regard to cluster of differentiation 44 (CD44) were investigated. The SGC-7901 cells were exposed to various concentrations of IL-8 (0, 0.2, 0.5, 0.8 and 1 ng/ml) in vitro. The adhesion of the SGC-7901 cells to fibronectin, an extracellular matrix component, was then detected by cell counting kit 8 assay. Migration and invasion abilities were evaluated by wound scratch and Transwell chamber assays. In addition, protein and mRNA levels of CD44 were measured using immunofluorescence and western blotting, and quantitative polymerase chain reaction, respectively, in cells cultured for 72 h. Following the exposure of the SGC-7901 cells to the various low doses of IL-8, the cell adhesion, migration and invasion capacities were promoted by IL-8, but not in a significant dose-dependent manner. Low-dose IL-8 upregulated the protein and mRNA expression of CD44. In conclusion, low-dose IL-8 potently induces the adhesion, migration and invasion of SGC-7901 cells, and the regulation of CD44 expression is one of the potential molecular mechanisms involved.

Abstract 509: Hypoxia and HIF silencing mediated dysregulation of total choline, CD44 expression, and metastatic burden in MDA-MB-231 human breast cancers

Abstract Hypoxic tumors frequently exhibit an aggressive phenotype due to dysregulated gene expression and metabolic changes. Hypoxia results in the stabilization of hypoxia inducible factors (HIF-1/2) that transcriptionally activate genes involved in invasion, metastasis, metabolism, and in the adaptation of cancer cells to their microenvironment. In breast cancer, stem-like breast cancer cells that survive, repopulate and metastasize to distant locations, have elevated expression of CD44. In a previous study, we observed elevated expression of CD44 in hypoxic tumor regions, and identified HIF-1α as a regulator of CD44 expression in breast cancer cells under hypoxic conditions [1]. Hypoxia has also been implicated in increasing the activity of choline kinase (Chk)-alpha, the enzyme responsible for elevated phosphocholine (PC) and total choline (tCho) consistently observed in cancers [2]. In previous studies, lentiviral transduction of MDA-MB-231 breast cancer cells (231 cells) with shRNA against Chk-alpha and the in vivo delivery of the Chk-shRNA virus into tumor bearing mice resulted in decreased CD44 message and expression together with effective silencing of Chk message and expression [3]. Here, using non-invasive proton magnetic resonance spectroscopic imaging (1H MRSI), we have established the importance of HIF in reducing total choline and metastatic tumor burden, and have identified a role for CD44 in establishing lung metastasis. HIF silencing in MDA-MB-231 cells significantly delayed tumor growth in mice. Both, the in vitro 1H and 31P MR spectra and in vivo 1H MRS images of tumors derived from engineered cells showed decreased tCho levels and distribution. This decrease of tCho was statistically significant in tumors derived from double silenced cells. Western blot analysis of tumors detected a decrease in Chk expression in double silenced (HIF-1 and 2) tumors. Silencing HIF-1α, -2α or both resulted in a significant reduction of metastatic lung burden in mice. Additionally, HIF-2α silencing was more effective at reducing lung colonization than HIF-1α, while silencing both was the most effective. Although metastatic burden decreased in HIF-1α silenced cells, the percentage of cells with high CD44 expression in the metastatic foci was comparable to that in the wild type or empty vector foci. These data identify the importance of targeting HIF and CD44 to prevent lung colonization and disrupt the metastatic cascade. This work was supported by NIH R01CA136576 and P50 CA103175. We thank Mr. Gary Cromwell for valuable technical assistance.

Abstract 3598: Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in gastrointestinal cancer cells and promote redox adaptation

Abstract Background: CD44 has recently been identified as one of the cell surface markers associated with cancer stem cells (CSCs) in several types of tumor. We have reported the functional role of CD44 variant isoform in the maintenance of low reactive oxygen species (ROS) levels in gastrointestinal cancer cells. While, microRNA (miRNA) is involved in the pathogenesis of many cancers and the networks of miRNA regulate CSCs properties. However, the mechanism underlying the miRNA in the regulation of CSC marker is mostly unrevealed. Objectives: The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop newmolecular targets in gastrointestinal cancer. Methods and Results: To investigate the mechanism in the regulation of CD44, We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 down-regulation and CD44 up-regulation in 63 patients with surgically-resected gastric cancer. Conclusions: Macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer. Citation Format: Takatsugu Ishimoto, Naoya Yoshida, Hidetaka Sugihara, Daisuke Izumi, Keisuke Miyake, Hiroshi Sawayama, Yu Imamura, Shiro Iwagami, Yoshifumi Baba, Hideo Baba. Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in gastrointestinal cancer cells and promote redox adaptation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3598. doi:10.1158/1538-7445.AM2014-3598

Regulation of CD44 expression by tumor necrosis factor-α and its potential role in breast cancer cell migration

CD44 molecule plays critical role in distant malignant metastasis. It is expressed in standard form (CD44s) or variant form (CD44v). Tumor necrosis factor-α (TNF-α) is highly expressed in the cancer microenvironment. TNF-α was reported to modulate CD44 expression in several kinds of cancer. However, little is known about pathological role of TNF-α in breast cancer (BC) cells. In the current investigation, we investigated the effect of TNF-α on BC cells (MCF-7 and MDA-MB-231) viability, CD44 expression, and in vitro migration. We found that TNF-α down-regulated CD44s expression, up-regulated CD44v3 and CD44v6 expression through JNK pathway in MCF-7 cells. In MDA-MB-231 cells, TNF-α up-regulated CD44s, CD44v3 and CD44v6 expression via p38 pathway. These data indicate important role of CD44 molecule in BC pathology.

A Novel Gemini Vitamin D Analog Represses the Expression of a Stem Cell Marker CD44 in Breast Cancer

CD44 is a multifunctional transmembrane protein involved in cell proliferation, angiogenesis, invasion, and metastasis. CD44 is identified as a cancer stem cell marker, and the CD44-positive breast cancer cells are enriched in residual breast cancer cell populations after conventional therapies, suggesting that CD44 may be an important target for cancer prevention and therapy. Therefore, we investigated for the inhibitory effect of a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), on mammary tumor growth and CD44 expression in MCF10DCIS.com human breast cancer in vitro and in vivo. MCF10DCIS.com cells were injected into mammary fat pads in immunodeficient mice, and BXL0124 was then administered intraperitoneally (0.1 μg/kg body weight) or orally (0.03 or 0.1 μg/kg body weight) 6 days a week for 5 weeks. At necropsy, mammary tumors and blood were collected for evaluating tumor growth, CD44 expression, and serum calcium level. BXL0124 suppressed mammary tumor growth and markedly decreased the expression of CD44 protein in MCF10DCIS xenograft tumors without causing hypercalcemic toxicity. BXL0124 also inhibited the expression of CD44 protein and mRNA as well as the transcriptional activity of the CD44 promoter in cultured MCF10DCIS.com cells. The repression of CD44 expression induced by BXL0124 was blocked by siRNA vitamin D receptor (VDR), indicating that the regulation of CD44 expression by BXL0124 is a VDR-dependent event. The novel Gemini vitamin D analog, BXL0124, represses CD44 expression in MCF10DCIS.com cells in vitro and in xenograft tumors, suggesting an inhibitory role of a Gemini vitamin D derivative on breast cancer stem cells.

Abstract 5138: The regulation of CD44 expression in malignant brain tumor cells

Abstract Glioblastoma multiforme (GBM) is a highly aggressive brain tumor that demonstrates a high growth rate and the capacity to invade surrounding brain tissue. These characteristics result in a dismal prognosis for GBM patients. Current treatment options for GBM tumors involve surgical resection followed by radiation and/or chemotherapy. Disappointingly, GBM tumors are or become resistant to radiation and chemotherapy, and the invasiveness of GBM precludes curative removal of the tumor. GBM cells infiltration has been associated with the transmembrane glycoprotein, CD44, which is the principle cell surface receptor for hyaluronic acid (HA). The molecular mechanisms that regulate CD44 in GBM are not fully understood. However, CD44 protein expression has been associated with an AP-1 transcription factor, fos related antigen 1 (Fra-1), in another aggressive cancer, mesothelioma. Therefore, we investigated the regulation of CD44 expression in human glioma cells by Fra-1. Human GBM cell lines, U-251 MG, U-1242 MG, and A-172, were exposed to either epidermal growth factor (EGF, 20 ng/ml) or hepatocyte growth factor (HGF, 50 ng/ml) at varying time points and Western blot analysis was used to determine the protein expression of CD44 and Fra-1. CD44 and Fra-1 expression levels increased with longer exposures (4 to 24 hr) to EGF and HGF. To determine whether Fra-1 takes part in the regulation of CD44 expression, we utilized transient gene knockdown using siRNA directed against fra-1. fra-1-siRNA decreased Fra-1 expression in EGF- and HGF-treated GBM cells. Following EGF stimulation and fra-1-siRNA treatments, CD44 expression also decreased. However, HGF stimulation of fra-1-siRNA treated GBM cells showed no change in CD44 expression. These data suggest that Fra-1 regulation of CD44 expression is growth factor-specific. To investigate whether CD44-mediated cell adhesion is promoted by the expression of Fra-1, we analyzed GBM cell adhesion to HA. U-251 MG and U-1242 MG cell lines demonstrated an increase adherence to HA after growth factor simulation, 4hr and 24 hr respectively. This data is comparable to the initial western blot analysis of the GBM cells after growth factor simulation. However, A-172 cells did not adhere to HA regardless of growth factor stimulation. These data suggest that CD44 binding to HA is cell line specific. Taken together, our results suggest that CD44-mediated HA binding of GBM cells can be regulated by Fra-1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5138.

Abstract 644: A novel Gemini vitamin D analogue represses the expression of a stem cell marker CD44 in breast cancer

Abstract Breast cancer stem cells are defined as cancer cells with self-renewal capacity. Cancer stem cells are resistant to current chemotherapy and radiation, making it difficult to investigate the mechanisms of resistance and the therapeutic targets of the tumor initiating cell population. Recently, CD44 has been recognized as a useful cell-surface marker for detecting breast cancer stem cells, and the CD44+/CD24- phenotype selects a population with stem cell properties similar to basal-like cancer cells. CD44 is a multi-functional transmembrane protein involved in cell proliferation, angiogenesis, invasion and metastasis. The CD44 positive subpopulation of breast cancer cells are enriched in residual breast cancer cell populations after conventional therapies, suggesting that CD44 positive cells may be responsible for the recurrence of cancer and therefore it may be an important therapeutic target. We have previously shown that novel Gemini vitamin D analogs inhibit the proliferation of breast cancer epithelial cells and suppress tumorigenesis in different animal models of breast cancer. Since therapies targeting CD44 by vitamin D have not been investigated, we examined whether a novel Gemini vitamin D analog regulates CD44 expression in cultured human breast cancer cells or in mammary tumors. In basal-like MCF10DCIS.com human breast cancer cells, the Gemini vitamin D analog BXL0124 significantly inhibited the expression of CD44 protein and reduced the transcriptional activity of the CD44 promoter in vitro. To evaluate the inhibitory effects of Gemini vitamin D on CD44 expression in vivo, MCF10DCIS.com cells were xenografted into immunodeficient mice. When administered intraperitoneally (0.1 microgram/kg body weight) or orally (0.3 microgram/kg body weight) once a day for 6 weeks, Gemini vitamin D BXL0124 inhibited tumor growth and markedly decreased the expression of CD44 in the xenograft tumors. To determine whether the repression of CD44 is dependent on the vitamin D receptor (VDR), siRNA VDR was employed. The repression of CD44 expression by BXL0124 was significantly blocked by siRNA VDR in MCF10DCIS.com cells, indicating that the regulation of CD44 expression by BXL0124 is a VDR-dependent event. Taken together, the novel Gemini vitamin D analog inhibits CD44 expression in MCF10DCIS.com cells in vitro and in xenograft tumors, suggesting an inhibitory role of Gemini vitamin D analogs on cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 644.

Value of CD44 immunostaining (IHC) as a surrogate in the differentiation of MYC-positive Burkitt lymphoma (BL) and Burkitt lymphoma-like (BLL) from MYC-negative Diffuse Large B-Cell Lymphoma (DLBCL) in a resource constrained clinical setting

Background Both BL and DLBCL are prevalent in HIV/AIDS patients and the correct classification of these two subtypes of Non-Hodgkin's lymphomas (NHL) particularly between DLCBL with high proliferation and BL, is critical to chemotherapy choice. Transcriptional gene expression profiling of sporadic and endemic BL by Stein et al (Berlin) identified a molecular signature for BL that included a down regulation of CD44 expression in a group of lymphomas from Africa. Immunophenotyping of formalin-fixed paraffin-embedded (FFPE) BL tissues by this group demonstrated difficulties in correctly classifying BL using morphology, BCL-2 (immunohistochemistry – IHC) presence and MYC (fluorescent in situ hybridization – FISH) absence. The differential expression of CD44, albeit nonmembranous, suggests an intricate regulatory circuit that allows for low expression, supporting the possibility that CD44 plays a role in BL/BLL dissemination. CD44 (IHC) could be a valuable marker to aid classification of tumors whose morphologic and phenotypic overlap is among BL, BLL and DLBCL. CD44 absence clearly separates MYC+ BL/BLL and MYC-DLBCL. DLBCL, Hodgkin's lymphoma (HL) and normal lymphocytes from lymphoid mantle/ marginal cell and interfollicular T-cell-rich zones should be strongly CD44 positive. Materials and methods We evaluated 128 FFPE lymphoma tissue biopsy collected at St. Mary's Lacor Hospital, Gulu, Uganda, Africa, from 1994 to 2007 from tissue microarray (TMA) blocks constructed on site using the UNITMA Quick-Ray System (Seoul, South Korea). The completed TMA blocks were sectioned and stained for HE in situ hybridization (ISH) for EBV (EBER) and kappa and lambda light chains and FISH break apart assay for CMYC translocation.

Differential binding of hyaluronan on the surface of tissue-specific endothelial cell lines.

Tissue-specific heterogeneity of endothelial cells, both structural and functional, plays a crucial role in physiologic as well as pathologic processes, including inflammation, autoimmune diseases and tumor metastasis. This heterogeneity primarily results from the differential expression of adhesion molecules that are involved in the interactions between endothelium and circulating immune cells or disseminating tumor cells. Among these molecules present on endothelial cells is hyaluronan (HA), a glycosaminoglycan that contributes to primary (rolling) interactions through binding to its main receptor CD44 expressed on leukocytes and tumor cells. While the regulation of CD44 expression and function on either leukocytes or tumor cells has been well characterized, much less is known about the ability of endothelial cells to express HA on their surface. Therefore, in these studies we analyzed HA levels on tissue-specific endothelium. We used endothelial cell lines of different origin, including lung, skin, gut and lymph nodes that had been established previously as model lines to study interactions between the endothelium and leukocytes/tumor cells. Our results indicate that HA is accumulated on the surface of all endothelial cells examined. Moreover, retention of endogenous HA differs between the lines and may depend on their tissue origin. Analysis of binding of exogenous HA reveals the presence of specific HA binding sites on all endothelial cell lines tested. However, the retention of endogenous HA and the binding of exogenous HA is mediated through a CD44-independent mechanism.

STAT6 is a novel regulator of CD44 expression in human B cells (97.4)

Abstract Interactions of CD44, an adhesion molecule, with its ligand, hyaluronan (HA) play a crucial role in cell migration, inflammation and immune response. The regulation of CD44 expression particularly in human B cells is not well understood. We have previously demonstrated that CD44 expression in human monocytic cells is regulated by Egr-1 and AP-1 transcription factors (Mishra et al. 2005, JBC). IL-4, a pleotropic cytokine is known to regulate CD44-HA interactions, cell migration, and differentiation of human B cells. To understand the regulation of IL-4-induced CD44 expression in human B cells, we used an EBV-transformed Burkitt’s lymphoma cell line, BL30/B95-8 as a model system. Our results suggested that IL-4 did not induce the expression of Egr-1 and AP-1 as determined by Northern and Western blot analysis and gel shift assays. To elucidate the transcription factors involved, we demonstrated by promoter analysis and gel shift assays that STAT-6 plays a critical role in IL-4-induced CD44 regulation in human B cells. We also investigated the upstream signaling events and demonstrated that IL-4-induced STAT-6 is activated by two distinct signaling pathways namely Jak-1/3 and ERK-MAPK and is independent of the IRS2/PI3 kinase pathway. Taken together, our results suggest a novel pathway of CD44 regulation involving the activation of STAT-6 through the upstream Jak-1/3 and ERK-MAPK in IL-4-stimulated human B cells.

Identification of novel splice variants of the human CD44 gene

The CD44 gene contains 10 variable exons (v1–v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms, several of which have been implicated in the metastatic spread of tumour cells. Here, we describe a cryptic splice site, in intron 6 of the human CD44 gene, used during mRNA processing. This cryptic splice site is used in conjunction with variable exon 3, or independently from it in the form of a pseudo-exon of 49 bp, which generates a stop codon by frame shift in the contiguous variable exon downstream. This pseudo-exon has been found inserted immediately 3′ to any other variable exon from v4 to v10, in the final CD44 mRNA. The implication of this cryptic splice site in haltering CD44 protein translation is questioned in the context of Nonsense Mediated Decay and the overall regulation of CD44 expression.

Expression of L-Selectin (CD62L), CD44, and CD25 on activated bovine T cells.

Mycobacterium bovis infection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4(+) cells from infected animals. CD62L MFI on PPD- and PWM-stimulated gammadelta T-cell receptor-positive (TCR(+)) and CD8(+) cells was also reduced compared to that of nonstimulated gammadelta TCR(+) and CD8(+) cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4(+) cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.

Cytokine Regulation of CD44 Expression on rat Intestinal Epithelial Cells

CD44 comprises a family of type I transmembrane glycoproteins that is expressed on a wide range of cells including those of epithelial, lymphoid and myeloid lineage. Although expression of CD44 in the small intestine is typically localised in the crypts of Lieberkühn, we have reported the expression of CD44 on mature, intestinal villus epithelial cells during the development of small bowel allograft rejection. The mechanisms underlying CD44 up-regulation are unknown, although it may be influenced by localised cytokine production. This study used flow cytometry to assess the effects of recombinant IFN-γ and TNF-α on CD44 expression and hyaluronan binding by the rat small intestinal epithelial cell lines, RIE and IEC 6. IFN-γ upregulated CD44 expression on RIE (155% of unstimulated control) and IEC 6 (209% of unstimulated control) cells, whereas TNF-α had no effect. IFN-γ had no qualitative effect on CD44, as binding of the ubiquitously expressed extracellular matrix polysaccharide hyaluronan was unchanged. RIE and IEC 6 cells expressed the 82 kDa and 130 kDa major isoforms of CD44, however cytokine stimulation did not affect the expression of these, nor did stimulation induce the expression of other variants. In summary, these findings demonstrate that CD44 expression by intestinal epithelial cells can be regulated by cytokines, yet their ability to bind hyaluronan and the isoform of the expressed CD44 remains unaltered. It appears that localised inflammatory conditions and cytokine production may modify epithelial cell expression of CD44, however the physiological role for such a response has yet to be elucidated.