Down-regulation Of CD44 Expression Research Articles

Ezrin Inhibition Overcomes Acquired Resistance to Vemurafenib in BRAFV600E-Mutated Colon Cancer and Melanoma Cells In Vitro.

Despite the advancements in targeted therapy for BRAFV600E-mutated metastatic colorectal cancer (mCRC), the development of resistance to BRAFV600E inhibition limits the response rate and durability of the treatment. Better understanding of the resistance mechanisms to BRAF inhibitors will facilitate the design of novel pharmacological strategies for BRAF-mutated mCRC. The aim of this study was to identify novel protein candidates involved in acquired resistance to BRAFV600E inhibitor vemurafenib in BRAFV600E-mutated colon cancer cells using an integrated proteomics approach. Bioinformatic analysis of obtained proteomics data indicated actin-cytoskeleton linker protein ezrin as a highly ranked protein significantly associated with vemurafenib resistance whose overexpression in the resistant cells was additionally confirmed at the gene and protein level. Ezrin inhibition by NSC305787 increased anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant cells in an additive manner, which was accompanied by downregulation of CD44 expression and inhibition of AKT/c-Myc activities. We also detected an increased ezrin expression in vemurafenib-resistant melanoma cells harbouring the BRAFV600E mutation. Importantly, ezrin inhibition potentiated anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant melanoma cells in a synergistic manner. Altogether, our study suggests a role of ezrin in acquired resistance to vemurafenib in colon cancer and melanoma cells carrying the BRAFV600E mutation and supports further pre-clinical and clinical studies to explore the benefits of combined BRAF inhibitors and actin-targeting drugs as a potential therapeutic approach for BRAFV600E-mutated cancers.

Knockdown of ALDH1A3 reduces breast cancer stem cell marker CD44 via the miR-7-TGFBR2-Smad3-CD44 regulatory axis.

Inhibition of aldehyde dehydrogenase 1 family member A3 (ALDH1A3) has been revealed to lead to significant increase of microRNA (miR)-7 expression and decrease of CD44 expression in breast cancer stem cells (BCSCs), however the mechanism is not clear. The aim of the present study was to investigate the regulatory relationship between ALDH1A3, miR-7, and CD44 in BCSCs. The expression of ALDH1A3 was inhibited by small interfering RNA (siRNA or si), and the expression of miR-7 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Then, the ratio of CD44+ cells was analyzed by flow cytometry in MDA-MB-231 cells. The dual-luciferase reporter system was used to demonstrate that miR-7 binds to transforming growth factor-β receptor 2 (TGFBR2) 3'UTR, and ChIP-PCR determined whether the transcription factor Smad3 binds to the upstream regulatory region of the CD44 promoter. The results revealed that siALDH1A3 downregulated ALDH1A3 and promoted miR-7 expression, which resulted in downregulation of CD44 expression. siALDH1A3 also downregulated the CD44 expression on the surface of MDA-MB-231 cells and inhibited the G2/M phase in BCSCs as analyzed by flow cytometry. In addition, lenti-miR-7 cells transfected with TGF-β1 + SB431542 revealed that lenti-miR-7 inhibited the TGF-β1 pathway by inhibiting Smad2/3/4 expression and, thus, downregulated CD44 expression. miR-7 was revealed to directly bind to the TGFBR2 3'UTR through dual-luciferase reporter assay, and Smad3, a transcription factor, through ChIP-PCR was demonstrated to bind to the upstream region of the CD44 promoter. These results demonstrated the existence of the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 axis in MDA-MB-231 cells. RT-qPCR results of 12 breast cancer surgical specimens and SK-BR-3, MCF-7, and LD cell lines further confirmed the presence of the regulatory axis. In conclusion the findings from the present study demonstrated that the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 regulatory axis was highly efficient in the inhibition of CD44 expression in BCSCs, and that the regulatory expression of ALDH1A3 and miR-7 may provide a strategy in the therapy of breast cancer.

CD44 Expression Predicts Prognosis of Ovarian Cancer Patients Through Promoting Epithelial-Mesenchymal Transition (EMT) by Regulating Snail, ZEB1, and Caveolin-1.

Objectives: CD44, a transmembrane glycoprotein, is involved in the generation of a stem cell niche and maintaining stem cell quiescence. The aim of this study was to evaluate its contribution to ovarian cancer prognosis and progression, as well as explore the possible mechanisms.Materials and Methods: The expression of CD44 in tissue microarray of 90 ovarian cancer patients was detected by immunohistochemistry. Kaplan-Meier method and Cox proportional hazard model were used to evaluate the factors associated with 5-year overall survival and disease-free survival. CD44 was knocked down by small interfering RNA, the expression of Snail, ZEB1, and Caveolin-1 in a stable Snail-expressing ovarian cancer cell line HO8910PM-Snail (HOPM-Snail) and its control cell line HO8910PM-vector (HOPM) was detected by western blotting analysis. Cell clone formation, migration, and invasion of HOPM-Snail and HOPM cells with CD44 silencing were examined by 3-D culture assay, wound healing assay, and transwell assay, respectively.Results: Over-expression of CD44 was associated with advanced histological grade (p = 0.014) and FIGO stage (p = 0.001). Multivariate analysis showed that CD44 expression was an independent prognostic factor to predict both overall survival (p = 0.004) and disease-free survival (p = 0.025) of ovarian cancer patients. Down-regulation of CD44 expression by small silencing RNA abrogated both basal Snail expression and TGF-β1-induced Snail expression in HOPM and HOPM-Snail cells. In addition, CD44 knockdown caused a decrease in ZEB1 expression. RPPA data indicated that Caveolin-1 may be another regulative target of CD44, and western blotting analysis confirmed that CD44 knockdown caused an increase in Caveolin-1 expression. However, there was no noticeable reciprocal regulation among ZEB1, Caveolin-1, and Snail. Moreover, CD44 knockdown caused a decrease in cell clone formation, migration, and invasion of HOPM and HOPM-Snail cells.Conclusions: As both Snail and ZEB1 are crucial inducers of epithelial-to-mesenchymal transition (EMT), our data suggested that CD44 may be crucial for the EMT process of ovarian cancer. Therefore, CD44 may be a potential prognostic marker as well as treatment target for ovarian cancer.

Cd20 Expression and Effects on Outcome of Relapsed/ Refractory Diffuse Large B Cell Lymphoma after Treatment with Rituximab

Introduction:Down regulation of CD20 expression has been reported in diffuse large B cell lymphoma (DLBCL)). Therefore, it is important to determine whether chemotherapy with rituximab induces CD20 down regulation and effects survival.Objectives:To determine the incidence of down regulation of CD20 expression in relapsed DLBCL after treatment with rituximab and to compare outcomes and assess pattern of relapse between CD20 negative and CD20 positive cases.Methodology:We retrospectively reviewed patients with relapsed DLBCL who received rituximab in the first line setting at Aga Khan University Hospital between January 2007 and December 2014. Data were recorded on predesigned questionnaires, with variables including demographics, details regarding date of diagnosis and relapse, histology, staging, international prognostic index, treatment and outcomes at initial diagnosis and at relapse. The Chi square test was applied to determine statistical significance between categorical variables. Survival curves were generated by the Kaplan–Meier method.Results:A total of 54 patients with relapsed DLBCL were included in our study, 38 (70 %) males and 16(30%) females. Some 23 (43%) patients were at stage IV at the time of diagnosis and 34 (63%) had B symptoms. The most frequent R-IPI at diagnosis was II in 24 (44%) patients. Only 6 (11%) did not show CD20 expression on re-biopsy for relapsed/refractory disease, 2 with CD20 negative DLBCL responding to second line chemotherapy. A complete response after salvage chemotherapy was noted in 16 (29.6%) cases with relapsed/refractory DLBCL. Seven (13%) patients underwent an autologous bone marrow transplant as consolidation after second line treatment. Median overall survival was 18 months in CD20 positive vs. 13 months in CD20 negative patients.Conclusion:This study demonstrated that a small percentage of patients treated with rituximab lose their CD20 expression at the time of relapse. However, it is unclear whether this is associated with an inferior outcome.

Effect of Arsenic Trioxide on K562 Cell Proliferation and Its Mechanism

To investigate the molecular mechanism of arsenic trioxide(ATO) inhibiting K562 cell proliferation, and explore the new targets for treating chronic myeloid leukemia(CML). human CML cell line K562 cells were cultured in vitro, and were treated with different concentrations of ATO; MTT was used to detect the cell proliferation; flow cytometry(FCM) was used to determine cell apoptosis, cell cycle and the expression of CD44; Transcriptional levels of β-catenin and cyclin D1 were assayed by RT-PCR. 2 µmol/L ATO could inhibit the cell proliferation obviously in a time-and-dose-dependent manner. With drug concentration increasing and time prolonging, the expression rate of CD44 was declined gradrually. FCM with AnnexinV/PI double staining showed that K562 cells were induced to apoptosis after exposure to 2.5-10 µmol/L ATO for 48 hours and in dose-dependent manner. Treating with different concentration ATO for 48 hours, cell ratio of G0/G1 phase increased and cell ratio in S phase decreased gradually. RT-PCR showed that the expression of β-catenin and CyclinD1 decreased with increasing of drug concentration. ATO in certain concentration range can inhibit K562 cell proliferation, and induce the cell apotosis, the mechanismin influencing the Wnt/β-catenin pathway may be the downregulation of CD44 expression, arresting K562 cells in G0/G1 phase, and affecting the gene transcription, thus inhibiting K562 cell proliferation.

P138 Modulation of CD20 expression on B cells by anti-CD20 antibodies

The purpose of this communication is to describe a case that illustrates the interference of in vivo anti-CD20 treatment on B cell crossmatch (B-XM) results. A first kidney transplant candidate, female, white, 31 years-old, with systemic lupus erythematosus, without previous pregnancies, with one blood transfusion more than 15 years ago, came to the laboratory (day 1) with the brother (D1) and the mother (D2) as potential donors (D). No HLA I or II antibodies were detected by Luminex-Single Antigen assay (One Lambda). XM results with DTT-treated serum: negative T-CDC-AHG-XM with D1 and D2; positive B-CDC-XM with D1 and D2; negative auto-B-CDC-XM; positive CDC-XM against B cells from eight healthy individuals (B-CDC-PRA). Day 42: XMs with D2 and auto-XM: same results. Day 68: the patient was listed for transplant with kidney from deceased donor and four serum samples were sent to the laboratory afterwards. Day 203: all six available sera were simultaneously tested in auto-T and -B-XM, by CDC and by flow cytometry (FC) with cells from day 203: all sera were negative in auto-T-XM; the sera from days 1 and 42, which had been negative in the auto-B-CDC-XM with cells from days 1 and 42, respectively, were positive with B cells from day 203 and also positive against 100% of the B cells from the panel. The serum from day 75 was positive in the auto-B-CDC-XM, auto-B-FC-XM and B-CDC-PRA, whereas the serum from day 88 was positive only in the auto-B-FC-XM. In contrast, sera from days 174 and 203 were negative in the auto-B-XM by CDC and FC, and in B-CDC-PRA. After all these tests, we found out that the patient had been treated with anti-CD20 antibodies (Rituximab) 83 days before the first tests and we concluded: all positive B-XMs could be explained by the presence of anti-CD20 in the serum; the negative auto-B-XM with the sera and cells from days 1 and 42 could be explained by a low, or lack of, CD20 expression in the cells of the patient caused by in vivo modulation by anti-CD20 antibodies. Down-regulation of CD20 expression in B-cell lymphoma after treatment with Rituximab is a well-documented phenomenon but, to the best of our knowledge, this is the first description of CD20 modulation in normal B cells.

Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells

Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

Rational Design of Polymeric Hybrid Micelles with Highly Tunable Properties to Co‐Deliver MicroRNA‐34a and Vismodegib for Melanoma Therapy

A polymeric hybrid micelle (PHM) system with highly tunable properties is reported to co‐deliver small molecule and nucleic acid drugs for cancer therapy; this system is structurally simple and easy‐to‐fabricate. The PHM consists of two amphiphilic diblock copolymers, polycaprolactone‐polyethylenimine (PCL‐PEI) and polycaprolactone‐polyethyleneglycol (PCL‐PEG). PHMs are rationally designed with different physicochemical properties by simply adjusting the ratio of the two diblock copolymers and the near neutral PHM‐2 containing a low ratio of PCL‐PEI achieves the optimal balance between high tumor distribution and subsequent cellular uptake after intravenous injection. Encapsulating Hedgehog (Hh) pathway inhibitor vismodegib (VIS) and microRNA‐34a (miR‐34a) into PHM‐2 generates the VIS/PHM‐2/34a co‐delivery system. VIS/PHM‐2/34a shows synergistic anticancer efficacy in murine B16F10‐CD44+ cells, a highly metastatic tumor model of melanoma. VIS/PHM‐2/34a synergistically attenuates the expression of CD44, a vital receptor indicating the metastasis of melanoma. Intriguingly, inhibiting Hh pathway by VIS is accompanied by downregulation of CD44 expression, revealing that Hh signaling might be an upstream regulator of CD44 expression in melanoma. Thus, co‐delivery of miR‐34a and VIS demonstrates great potential in cancer therapy, and PHM offers a structurally simple and highly tunable platform for the co‐delivery of small molecule and nucleic acid drugs in tumor combination therapy.

High-Affinity CD20-Specific T-Cell Receptors Suitable for Adoptive Immunotherapy in the Treatment of CD20low B-Cell Malignancies

Therapeutic monoclonal antibodies (mAb) such as Rituximab and Ofatumumab have demonstrated the clinical efficacy of targeting the B-cell restricted antigen CD20 for the treatment of B-cell lymphomas and leukemia. Although CD20 is also expressed on healthy B-cell cells which are depleted in the course of therapy, long-term B-cell aplasia is well manageable. However, non-responsive or refractory disease to CD20-targeted mAb treatment has been reported with various mechanisms of resistance: downregulation of CD20 expression, internalization of CD20:mAb complex, inhibition of complement-dependent cytotoxicity and absence of an effector cell repertoire in patients treated with chemotherapy prior to mAb infusion. Therefore, additional therapeutic strategies are required.T-cell receptor (TCR) gene transfer is an attractive strategy to equip T-cells with TCRs of defined antigen-specificity. Due to their high sensitivity for cognate antigen presented in HLA, TCRs can induce T-cell activation even when antigen expression is very low. However, the broad application of TCR-based adoptive immunotherapy directed against self-antigens such as CD20 is hampered by lack of an effective immune response against self-antigens. T-cells carrying high-affinity TCRs reactive to such self-antigens are deleted by negative selection during thymic development to prevent auto-reactivity. An attractive strategy to target self-antigens is to exploiting the immunogenicity of such antigens presented in the context of allogeneic HLA (alloHLA).Here, we used the CD20-derived peptide SLFLGILSV (CD20SLF) binding in HLA-A2 to isolate CD20-reactive T-cells carrying high-affinity TCRs. From peripheral blood mononuclear cells of HLA-A*0201 (HLA-A2)-negative healthy individuals CD8+ T-cells binding to peptide-HLA tetramers composed of CD20SLF bound to HLA-A2 were isolated and clonally expanded. Two high-avidity T-cell clones were identified specific for HLA-A2-bound CD20SLF. CD20-dependent recognition was demonstrated for both clones by transducing the CD20 gene in HLA-A2-positive cell lines which otherwise lack CD20 expression. Both CD20-specific T-cell clones efficiently recognized CD20-expressing HLA-A2-positive primary B-cell malignancies including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In addition, the CD20-specific T-cell clones were able to more efficiently recognize ALL cell-lines than CD20-specific mAbs. We demonstrated that on target cells with only very low CD20 surface expression, the CD20-specific T-cell clones could still efficiently recognize endogenously processed CD20-derived peptide in the context of HLA-A2. Furthermore, no recognition of HLA-A2-positive but CD20-negative cell subsets including CD34+hematopoietic progenitor cells, T-cells, immature and mature dendritic cells could be demonstrated. Additionally, recognition of HLA-A2-positive non-hematopoietic cells such as fibroblasts even under simulated inflamed conditions was absent.Transduction of the identified TCRs resulted in efficient expression of the introduced CD20-specific TCRs and conferred CD20-specificity onto recipient cells.In summary, we exploited the immunogenicity of alloHLA to raise high-avidity T-cells against self-antigens such as CD20. The identified CD20-specific T-cell clones efficiently recognized CD20-expressing primary ALL, CLL and MCL. These T-cells clones more efficiently recognized B-cell malignancies than CD20-targeted mAbs while no recognition of CD20-negative hematopoietic and non-hematopoietic cells was observed. Transduction of these CD20-specific TCRs conferred CD20-specificity onto recipient cells. These CD20-specific TCRs can be useful to treat patients with CD20low B-cell malignancies by administering TCR-engineered T cells with potent effector function. DisclosuresNo relevant conflicts of interest to declare.

SY12-4 - Molecular Mechanisms of Rituximab Resistance and Strategies to Overcome Them

ABSTRACT Rituximab is the first anti-CD20 chimeric-monoclonal antibody utilized for patients, and is now a key molecular targeting drug for CD20-positive B-cell lymphomas (BCL). Although combination chemotherapies with rituximab have surely improved the clinical outcomes of BCL patients, many clinicians recognize that relapse/refractory disease, which occurs in almost 50% of BCL patients, is still a major problem. Confirming the molecular mechanisms of rituximab resistance is crucial for further improvement of overall survival and progression-free survival in BCL patients. Several mechanisms of rituximab resistance have recently been reported as follows: (i) downregulation of CD20 expression after using rituximab, (ii) low expression level of CD20 at diagnosis, (iii) CD20 genetic mutations, (iv) aberrant expression of complement regulatory molecules (e.g. CD55, CD59) on tumor cells), (v) disruption of lipid raft formation, (vi) abnormality of signal transduction pathway, and (vii) Fc-gamma receptor polymorphisms of effector cells. To overcome these phenomena, several strategies are considered: (i) utilization of newer generation anti-CD20 antibodies (e.g. type I and II antibodies), (ii) new monoclonal antibodies for other targets (e.g. anti-CD22, -CD23, -CD79), and (iii) combination therapy with other molecular targeting drugs (e.g. demethylating and deacetylating agents, BH3 mimetics). In this session, recent progress in molecular analyses of resistant mechanisms and the strategies to overcome them will be summarized. Understanding these mechanisms may provide quite important information for not only improving clinical outcomes of BCL patients but also confirming the resistance mechanisms against newer monoclonal antibodies for BCL and solid tumors.

P2-01-14: High Level of CD44 Expression and Soluble CD44 Secretion Mediate Trastuzumab Resistance in HER2 Positive Breast Cancer.

Abstract Background: Overexpression of HER2 occurs in 15–25% of invasive breast cancer. Trastuzumab, a humanized monoclonal antibody targeting the extracellular domain of HER2, markedly improves disease-free and overall survival in patients with HER2−positive breast cancer. However, resistance to trastuzumab develops in most patients with HER2−overexpressing metastatic breast cancer. Breast cancer stem cells have been shown to express HER2, although its role in the development or maintenance of stem cells is not well characterized. CD44 is one of the proposed markers for breast cancer stem cells. We investigated the role of CD44 in acquired trastuzumab resistance in HER2 positive breast cancer. Methods: CD44 mRNA expression was measured by quantitative real-time PCR. The proportion of CD44 positive cells was measured by fluorescence-activated cell sorting (FACS). Soluble CD44 level was detected by enzyme-linked immunosorbent assay (ELISA). In vitro tumorigenicity was evaluated by soft agar colony formation assay. Cell invasion was tested by using a matrigel invasion assay. In vivo tumor growth was evaluated using a mammary fat pad xenograft mouse model. Results: CD44 mRNA expression, CD44 positive cell population by FACS, and CD44 secretion were significantly increased in both SKBR3 derived trastuzumab resistant SKBR3 clone 3 and BT474 derived trastuzumab resistant HR20 cells compared to parental, trastuzumab sensitive cells. Consistent with the increase in CD44, trastuzumab resistant cells showed an increase in soft agar colony formation and cell invasion, which were blocked by siRNA-induced downregulation of CD44 expression. Tumor growth and incidence were enhanced in mice injected with trastuzumab resistant HR20 cells compared to the mice injected with trastuzumab sensitive parental BT474 cells. Knock-down of CD44 expression by the transfection with CD44-specific siRNA suppressed in vivo tumor growth in the mice injected with trastuzumab resistant HR20 cells. Finally, we found high serum CD44 levels correlated with advanced tumor progression in HER2 positive breast cancer. Conclusion: Our findings indicate that high levels of CD44 mediate trastuzumab resistance in HER2 positive breast cancer cells as well as tumor progression in mice. Circulating CD44 is prognostic in patients with HER2 positive breast cancer. CD44 may be a therapeutic target for HER2 positive breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-14.

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.

Inhibition of CD44 Expression by Small Interfering RNA to Suppress the Growth and Metastasis of Ovarian Cancer Cells in Vitro and in Vivo

Since ovarian cancer cells express CD44, which causes very strong cell adhesion to peritoneal mesothelium and an unfavourable prognosis, we designed small interfering RNA (siRNA) targeting the CD44 gene to analyse the functional consequences of this inhibition in human ovarian cancer. We transfected ovarian cancer cell line SKOV-3 with well-designed CD44 siRNA or control siRNA. Western blot analysis was used to assess the CD44 expression. Following stable transfection, significant inhibition of CD44 expression with 66.13 ± 4.21 % (P < 0.05) in CD44 siRNA1 cells and 62.01 ± 3.97 % (P < 0.05) in CD44 siRNA2 cells was detected. We performed in vitro experiments including cellular adhesion to hyaluronan and human peritoneal mesothelial cells, etoposide-induced apoptosis, and Boyden chamber invasion assays. The adhesion percentages of CD44 siRNA1 and CD44 siRNA2 cells were significantly lower than those of the control siRNA cells in adhesion both to hyaluronan and to human peritoneal mesothelium. The CD44 siRNA transfectants showed significant inhibition of in vitro invasion and loss of resistance to apoptosis than the control siRNA cells. In vivo study with BALB/c mice was applied to compare the tumour growth and peritoneal dissemination. Nude mice treated with CD44 siRNA cells revealed significantly lower tumour volume and less peritoneal dissemination compared to mice treated with the control siRNA cells. In conclusion, down-regulation of CD44 expression by siRNA inhibits the in vitro adhesion, invasion and resistance to apoptosis of ovarian cancer cells, suppresses tumour growth and peritoneal dissemination of human ovarian cancer xenograft in nude mice.

Epigenetic Regulation of CD20 Protein Expression in B-Cell Lymphoma Cells after Rituximab-Containing Chemotherapy.

For the past 10 years, rituximab has been used with some forms of conventional chemotherapy to treat CD20(+) B-cell malignancies as a new molecular targeting drug. Although significant improvements in disease-free survival and cure rate have been obtained, resistance to rituximab has been recognized as a considerable problem. Here we report that CD20(−) tumor transformation is one important mechanism of resistance to rituximab. We also demonstrate a possibility that CD20 protein expression is downregulated by some epigenetic mechanisms, and interestingly, the protein expression can be restored by treatment with certain epigenetic drugs. During the 5 years beginning in 2001, 124 patients with B-cell malignancies were treated with rituximab-containing induction, consolidation, or salvage chemotherapies in our hospital. Relapse or recurrence of disease were confirmed in 36 patients (29.0%), and re-biopsy of tumor tissues was performed in 19 patients. Five cases (DLBCL, 3 cases, and DLBCL transformed from FL, 2 cases) of those 19 patients (26.3%) indicated CD20 protein-negative transformation in flow cytometry (FCM) and/or immunohistochemistry (IH) analyses. We established a CD20(−) lymphoma cell line RRBL1 from a DLBCL patient with CD20(−) transformation from CD20(+) FL after repeated treatment with rituximab, and analyzed molecular mechanisms of the CD20 protein-negative phenotype. CD20 was not expressed by IH, FCM, and immunoblotting analyses, and resistance to rituximab was observed after cell culture with rituximab treatment. Expression of wild-type CD20 mRNA was confirmed by RT-PCR, and no genetic mutation in coding sequence or promoter region was observed. Quantitative RT-PCR showed that the CD20 mRNA expression level was almost 100 times lower than that of CD20(+) B-cell lymphoma cells typically obtained from DLBCL patients. These data suggest that aberrant down regulation of CD20 mRNA may be closely related to the negative CD20 surface antigen expression. Interestingly, CD20 protein re-expression was confirmed by enhancing CD20 mRNA expression using specific epigenetic drugs including histone deacetylase inhibitor Trichostatin A (TSA). In addition, after TSA treatment, sensitivity for rituximab was significantly restored. Chromatin immunoprecipitation assay suggested that recruitment of histone deacetylase complexes to CD20 promoter may be related to the down regulation of CD20 expression. Our data may provide the basis for a new therapeutic strategy, epigenetic therapy combined with molecular targeting therapy using rituximab, for rituximab-resistant CD20(−) transformed B-cell malignancies.

Store-operated Cation Entry Mediated by CD20 in Membrane Rafts

B cell activation requires sustained elevation of cytoplasmic free calcium, achieved by influx through store-operated calcium (SOC) channels. The molecular identity of these channels is not known. Ectopic expression of the raft-associated tetraspan protein CD20 in Chinese hamster ovary cells introduced a novel SOC entry pathway that was permeable to strontium as well as to calcium. The activity of this SOC pathway was abolished by deletion of a cytoplasmic sequence in CD20 essential for its efficient raft localization. Strontium-permeable SOC channels were detected in B cells, and B cell receptor-stimulated influx was significantly reduced by downregulation of CD20 expression using short interfering RNA and also by cholesterol depletion. This is the first evidence that raft-associated CD20 constitutes a component of a SOC entry pathway activated by the B cell receptor.

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.

Down-regulation of CD44 expression in human prostatic carcinoma cell lines is correlated with DNA hypermethylation.

Down-regulation of the cell-surface adhesion molecule CD44 has been suggested to play an important role in tumor progression and metastasis of prostate cancer. CD44 is encoded by a gene that contains a CpG-rich region (CpG island) in its 5' regulatory sequence. We tried to assess whether hypermethylation of this region is the mechanism responsible for CD44 transcriptional inactivation. A panel of prostatic-carcinoma cell lines, Du145, LNCaP, PC3, PC346C and TSU, was analyzed for CD44 mRNA and protein expression. Du145, PC3 and TSU were positive for CD44, whereas in LNCaP and PC346C both CD44 mRNA and protein expression was suppressed. Methylation-sensitive restriction-enzyme analysis of genomic DNA showed that, in contrast to the CD44-positive cell lines, the CD44-negative lines were hypermethylated in the CD44 promoter CpG island. Furthermore, treatment of a PC346C culture with the demethylating agent 5-azacytidine resulted in re-expression of CD44 mRNA. It is concluded that hypermethylation of the CD44 5' promoter region is one of the mechanisms by which CD44 expression is down-regulated in prostatic-carcinoma cell lines.