Adhesion Receptor CD44 Research Articles

TDO2 modulates liver cancer cell migration and invasion via the Wnt5a pathway.

Liver cancer is a malignant cancer phenotype for which there currently remains a lack of reliable biomarkers and therapeutic targets for disease management. Tryptophan 2,3‑dioxygenase(TDO2), a heme‑containing polyoxygenase enzyme, is primarily expressed in cells of the liver and nervous systems. In the present study, through the combination of cancer bioinformatics and analysis of clinical patient samples, it was shown that TDO2 expression in liver cancer tissue samples was significantly higher than that in normal tissues, and liver cancer patients with high TDO2 expression had a poor prognosis. Mechanistic studies on liver cancer cells showed that TDO2 promoted cancer cell migration and invasion via signal transduction through the Wnt5a pathway. Such regulation impacted the expression of cancer‑associated biomarkers, such as matrix metalloprotease 7 (MMP7) and the cell adhesion receptor CD44. Treatment with a calcium channel blocker (azelnidipine) reduced TDO2 levels and inhibited liver cancer cell migration and invasion. A mouse xenograft cancer model showed that TDO2 promoted tumorigenesis. Furthermore, azelnidipine treatment to downregulate TDO2 also decreased liver cancer development in this mouse cancer model. TDO2 is thus not only a useful liver cancer biomarker but a potential drug target for management of liver cancer.

Evaluation of lumican effects on morphology of invading breast cancer cells, expression of integrins and downstream signaling.

The small leucine-rich proteoglycan lumican regulates estrogen receptors (ERs)-associated functional properties of breast cancer cells, expression of matrix macromolecules, and epithelial-to-mesenchymal transition. However, it is not known whether the ER-dependent lumican effects on breast cancer cells are related to the expression of integrins and their intracellular signaling pathways. Here, we analyzed the effects of lumican in three breast cancer cell lines: the highly metastatic ERβ-positive MDA-MB-231, cells with the respective ERβ-suppressed (shERβMDA-MB-231), and lowly invasive ERα-positive MCF-7/c breast cancer cells. Scanning electron microscopy, confocal microscopy, real-time PCR, western blot, and cell adhesion assays were performed. Lumican effects on breast cancer cell morphology were also investigated in 3-dimensional collagen cultures. Lumican treatment induced cell-cell contacts and cell grouping and inhibited microvesicles and microvilli formation. The expression of the cell surface adhesion receptor CD44, its isoform and variants, hyaluronan (HA), and HA synthases was also investigated. Lumican inhibited the expression of CD44 and HA synthases, and its effect on cell adhesion revealed a major role of α1, α2, α3, αVβ3, and αVβ5 integrins in MDA-MB-231 cells, but not in MCF-7/c cells. Lumican upregulated the expression of α2 and β1 integrin subunits both in MDA-MB-231 and in shERβMDA-MB-231 as compared to MCF-7/c cells. Downstream signaling pathways for integrins, such as FAK, ERK 1/2 MAPK 42/44, and Akt, were found to be downregulated by lumican. Our data shed light to the molecular mechanisms responsible for the anticancer activity of lumican in invasive breast cancer.

CD44 Promotes PD-L1 Expression and Its Tumor-Intrinsic Function in Breast and Lung Cancers.

The PD-L1 (CD274) immune-checkpoint ligand is often upregulated in cancers to inhibit T cells and elicit immunosuppression. Independent of this activity, PD-L1 has recently been shown to also exert a cancer cell-intrinsic function promoting tumorigenesis. Here, we establish this tumor-intrinsic role of PD-L1 in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). Using FACS-assisted shRNA screens, we identified the cell-surface adhesion receptor CD44 as a key positive regulator of PD-L1 expression in these cancers. Mechanistically, CD44 activated PD-L1 transcription in part through its cleaved intracytoplasmic domain (ICD), which bound to a regulatory region of the PD-L1 locus containing a consensus CD44-ICD binding site. Supporting this genetic interaction, CD44 positively correlated with PD-L1 expression at the mRNA and protein levels in primary tumor samples of TNBC and NSCLC patients. These data provide a novel basis for CD44 as a critical therapeutic target to suppress PD-L1 tumor-intrinsic function. SIGNIFICANCE: CD44 is a potential target to suppress PD-L1 function in TNBC. This finding has the potential to open a new area of therapy for TNBC.

Association of CD44 gene rs187115 polymorphism with colorectal cancer risk and prognosis in Chinese Han population: a case-control study.

The cell surface adhesion receptor CD44 reportedly affects the development and progression of cancers. Moreover, CD44 gene rs187115 polymorphism appears to be genetic determinant of cancer susceptibility. We investigated whether CD44 rs187115 polymorphism is associated with colorectal cancer (CRC) risk and prognosis. We enrolled 669 CRC cases and 826 controls in this three-center case-control study in a Chinese Han population. All individuals were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Cross-over analysis, multivariate logistic regression, Kaplan-Meier method, and Cox regression analysis were used for analysis. In this study, CD44 rs187115 polymorphism was associated with increased risk for CRC. Stratified analyses revealed that CD44 rs187115 polymorphism was correlated with increased risk for CRC in females, drinkers, smokers, and those aged ≥ 60 years. In addition, rs187115 polymorphism was significantly associated with TNM III+IV stage, lymph node metastasis and tumor size in CRC patients. Combined effects of CD44 rs187115 polymorphism (GG/AG vs. AA) and environmental factors (smoking and drinking) further increased the risk of CRC. GG genotype carriers showed poorer overall survival than AA genotype carriers. Cox regression analysis showed that drinking, CD44 rs187115 polymorphism, and TNM stage were independent prognostic factors affecting CRC. These findings show that CD44 rs187115 polymorphism may be a potential biomarker predictive of CRC susceptibility and prognosis.

A Three-Dimensional In Vitro Coculture Model to Quantify Breast Epithelial Cell Adhesion to Endothelial Cells.

Three-dimensional (3D) in vitro culture models better recapitulate the tissue microenvironment, and therefore may provide a better platform to evaluate therapeutic effects on adhesive cell-cell interactions. The objective of this study was to determine if AD-01, a peptide derivative of FK506-binding protein like that is reported to bind to the adhesion receptor CD44, would induce a greater reduction in breast epithelial spheroid adhesion to endothelial tube-like networks in our 3D coculture model system compared to two-dimensional (2D) culture. MCF10A, MCF10A-NeuN, MDA-MB-231, and MCF7 breast epithelial cells were pretreated with AD-01 either as single cells or as spheroids. Breast epithelial cell adhesion to 2D tissue culture substrates was first measured, followed by spheroid formation (breast cell-cell adhesion) and spheroid adhesion to Matrigel or endothelial networks. Finally, CD44 expression was quantified in breast epithelial cells in 2D and 3D culture. Our results show that AD-01 had the largest effect on spheroid formation, specifically in breast cancer cell lines. AD-01 also inhibited breast cancer spheroid adhesion to and migration along endothelial networks. The different breast epithelial cell lines expressed more CD44 when cultured as 3D spheroids, but this did not universally translate into higher protein levels. This study shows that 3D coculture models can enable unique insights into cell adhesion, migration, and cell-cell interactions, thereby enhancing understanding of basic biological mechanisms. Furthermore, such 3D coculture systems may also represent a more relevant testing platform for understanding the mechanism-of-action of new therapeutic agents. Impact Statement Cell adhesion is inherently different in two dimensional (2D) compared to three dimensional (3D) culture; yet, most adhesion assays in academia and industry are still conducted in 2D because few simple, yet effective, adhesion models exist in 3D. Recently we developed a 3D in vitro coculture model to examine breast epithelial spheroid interactions with endothelial tubes. We now show that this 3D coculture model can effectively be used to interrogate and quantify drug-induced differences in breast epithelial cell adhesion that are unique to 3D cocultures. This 3D coculture adhesion model can furthermore be modified for use with other cell types to better predict drug effects on cell-vasculature adhesion.

Polymeric nanoparticle-mediated silencing of CD44 receptor in CD34+ acute myeloid leukemia cells

The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem/progenitor cells (LSPC) within the bone marrow (BM) niche, as well as in the high relapse rates of acute myeloid leukemia (AML). Down-regulating CD44 could be clinically relevant not only for suppression of the deregulated function of LSPC but also in LSPC response to chemotherapeutic agents. Small interfering RNA (siRNA) delivery is a promising approach for AML treatment, and we recently reported effective siRNA delivery into difficult-to-transfect AML cell lines using lipid-substituted polyethylenimine/siRNA complexes (polymeric nanoparticles). In this study, we investigated polymeric nanoparticle-mediated silencing of CD44 in CD34+ LSPC cell models (leukemic KG-1 and KG-1a cell lines) as well as primary AML cells. Polymeric nanoparticle-mediated silencing decreased surface CD44 levels in KG-1, KG-1a and primary AML cells by up to 27%, 30% and 20% at day 3, respectively. Moreover, CD44 silencing resulted in induction of apoptosis in KG-1 cells, reduced adhesion of KG-1 and KG-1a cells to hyaluronic acid-coated cell culture plates and BM-MSC, and decreased adhesion of primary AML cells to BM-MSC. Our results suggest that polymeric nanoparticle-mediated silencing of CD44 might be a useful technique for inhibiting LSPC interactions with their microenvironment, thereby prohibiting leukemia progression or sensitizing LSPC to chemotherapy.

Early Changes of Gene Expression Profiles in the Rat Model of Arterial Injury

PurposeRestenosis caused by intimal hyperplasia (IH) remains a significant drawback for vascular interventions. It is crucial to understand the molecular mechanisms that control activation of smooth muscle cells (SMCs) after the injury in order to develop strategies to prevent IH. The purpose of the present study was to investigate the early alterations in arterial-wall gene expression after balloon injury in the rat carotid artery with focus on the induction of an inflammatory response. Materials and MethodsTwenty-four male Sprague–Dawley rats were subjected to injury of the left common carotid artery by using a 2-F Fogarty catheter. The arteries were harvested 5, 10, and 20 hours after injury. Uninjured arteries from an additional eight rats were used as controls. RNA was isolated and used for genome-wide microarray expression analysis, followed by validation of selected genes with quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed on the cross-sectioned vessels. ResultsAnalysis of gene expression by microarrays showed that the most differentially expressed genes were primarily associated with inflammation, cell proliferation, migration, and adhesion. As confirmed by qRT-PCR, microarray data showed a significant (P < .005) upregulation of cytokines and chemokines (IL-6, CCL2, CXCL1, AIMP1, and CD44) just 5 hours after injury. Immunohistochemistry demonstrated that CCL2 and the adhesion receptor CD44 were expressed by SMCs in the early response to injury and in the absence of leukocyte infiltration. ConclusionsArterial injury is followed by an early induction of inflammatory genes in the vessel wall that appears to be confined to SMCs.

Lysyl Oxidase at the Crossroads of Mesenchymal Stem Cells and Epithelial-Mesenchymal Transition

Lysyl Oxidase at the Crossroads of Mesenchymal Stem Cells and Epithelial-Mesenchymal Transition

Mti-101 (cyclized HYD1) Binds to CD44 and Induces Necrotic Cell Death in Multiple Myeloma

Abstract 4022Our laboratory has previously shown that treatment with the novel d-amino acid peptide HYD1 (kikmviswkg) induces necrotic cell death in multiple myeloma (MM) cell lines. Importantly, this novel peptide and similar compounds demonstrate increased activity in specimens obtained from relapsed patients (Nair et al. Mol Cancer Ther. 2009 Aug;8(8):2441-51., Emmons et al. Mol Cancer Ther. 2011 Dec;10(12):2257-66). Due to the intriguing biological activity of our linear peptide, we pursued strategies for increasing the therapeutic potential of this class of compounds. These efforts have lead to a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as single agent using two independent myeloma models which consider the bone marrow microenvironment. To further delineate the binding complex, we pursued an unbiased chemical biology approach using biotinylated peptide and LC-MS/MS analysis as an unbiased analytical tool for defining the HYD1 binding complex. Using this approach the adhesion receptor CD44 was determined to be among the adhesion receptors that had the most peptides identified and was confirmed via Western Blot analysis. Furthermore, we were able to show that HYD1 binds to recombinant CD44 through two independent binding assays. In preliminary ELISA-based binding experiments with full length recombinant human CD44, we were able to show that CD44 bound to biotin conjugated HYD1 in a concentration dependent manner. Additionally, we produced recombinant human CD44 ectodomain and utilizing surface plasmon resonance (SPR), we were able to show that the recombinant human CD44 ectodomain bound to biotin conjugated HYD1 and MTI-101 with a high affinity (Kd of approximately 30 nM). We are currently pursuing Isothermal Titration Calorimetry (ITC) binding assays to confirm binding affinities. Finally, using fluorescently tagged HYD1 (the 5(6)-FAM conjugate) as a molecular tool, we were able to demonstrate that overexpression of the standard form of CD44 (CD44s) in the 8226 myeloma cell line resulted in increased binding of our peptide. Paradoxically, increased expression of CD44s and subsequent increased binding of the FAM-conjugated peptide did not correlate with increased cell death. These data suggest that either CD44v expression is mediating cell death or that downstream or lateral signaling within the binding complex is rate limiting for cell death. Moreover, co-immunoprecipitation assays demonstrated that CD44 and the pro-necrotic receptor interacting protein 1 (RIP1) co-localized upon treatment with MTI-101 in NCI-H929 and U266 cells. Thus, we have elucidated a novel signaling pathway linking CD44 to RIP1, which is part of the necroptosome. Further studies using RNA interference are currently being conducted to determine the causative role of RIP1 and RIP3 in mediating HYD1 and MTI-101-induced necrotic cell death. Together, our data begin to elucidate the mechanism of action and definitively support continued development of MTI-101 for the treatment of relapsed myeloma, providing another therapeutic option for this currently incurable disease. Disclosures:McLaughlin:Modulation Therapeutics Inc: Patents & Royalties, Vice-President and Co-founder Other. Hazlehurst:Modulation Therapeutics Inc: Patents & Royalties, President and Co-founder Other.

Co-Expression of a Suicide Gene in CAR-Redirected T Cells Enables the Safe Targeting of CD44v6 for Leukemia and Myeloma Eradication

Abstract 949 Background:Acute myeloid leukemia (AML) and multiple myeloma (MM) share common features, such as preferential homing to the bone marrow (BM) and the tendency to relapse after chemotherapy. These phenomena are possibly linked, as signals provided within the BM niche induce chemoresistance. Relapsing AML and MM can however be cured by allogeneic hematopoietic stem cell transplantation (HSCT), suggesting that allospecific T may specifically sterilize tumor cells that survive chemotherapy. Unfortunaley, the universal application of HSCT is severely limited by the occurrence of life-threatening GVHD and by the fact that leukemia often escapes the alloimmune pressure by losing specific HLAs, as we have recently demonstrated (Vago et al., NEJM 2009). The isoform variant 6 of the adhesion receptor CD44 (CD44v6) is widely expressed in AML and MM, and is crucially involved in BM homing. In phase I/II trials, targeting CD44v6 with mAb conjugated to chemotherapeutic agents showed substantial efficacy, but also toxicity due to background expression of CD44v6 on the skin. Thanks to the chimeric antigen receptor (CAR) technology it is now possible to efficiently redirect T cells against virtually any antigen for which there is availability of a specific mAb. Our experience with suicide gene therapy in the context of HSCT (Ciceri et al., Lancet Oncol 2009) clearly indicates that genetic switches are efficient tools for controlling the off-tumor toxicities of T cells. Aim:To develop a strategy for the safe targeting of CD44v6 through the co-expression of a suicide gene in CAR-modified T cells for AML and MM treatment Results:CD44v6 surface expression was observed in 11/14 (79%) AML cases. Co-culturing primary leukemic cells with BM-derived mesenchymal stromal cells (MSCs) ex vivo caused the significant up-regulation of CD44v6 expression (P<0.05). At difference with AML, CD44v6 expression was constitutively high in the majority of MM cases (13/15, 87%). Upon MSC co-culturing, both primary leukemic cells and malignant plasma cells (mPCs) became resistant to cell death induced by daunorubicin (P<0.01) and bortezomib (P<0.05), respectively. Pre-incubating tumor cells with a CD44v6-blocking mAb reverted the chemoresistant phenotype. Importantly, knocking-down CD44v6 expression by (LV)-assisted short-hairpin (sh) RNA interference completely abated tumor-cell homing to the BM and tumorigenesis in NSG mice (P<0.001). We therefore constructed a CAR by cloning the scFv of a humanized CD44v6 mAb in a CD28/TCR zeta chain backbone and expressed it with the Herpes simplex virus thymidine kinase (HSV-tk) suicide gene by means of a LV carrying a PGK bi-directional promoter. After LV transduction, primary T cells concomitantly acquired potent and CD44v6-specific cytotoxicity against autologous leukemic cells and mPCs, and a selective sensitivity to the prodrug ganciclovir. Importantly, adding MSCs did not interfere with effector functions. Once infused into NSG mice, CD44v6-redirected T cells persisted long-term and completely eradicated previously engrafted autologous leukemic cells (P<0.005). Antitumor efficacy was dependent on transduction with CD3/CD28-beads and culture with IL-7/IL-15, according to a protocol that preserves the expression of the self-renewal marker IL-7 receptor and of the BM addressin CXCR4 (Bondanza, Blood 2011). Interestingly, CD44v6-redirected T cells were not cytotoxic against CD34+CD38- hematopoietic stem cells, but recognized mature monocytes. To overcome the problem of HSV-tk immunogenicity, we alternatively evaluated the novel, humanized suicide gene inducible caspase 9 (icasp9). Differently form HSV-tk, icasp9 activation by the specific chemical inducer of dimerization allowed the ablation of CD44v6-redirected T cells even in the absence of proliferation and with a much faster kinetics (<90% in 4 hrs vs 84 hrs, P<0.05) Conclusions:CD44v6 CAR-redirected suicidal T cells have the potential to eradicate AML and MM and can be ablated at will. We are currently designing a phase I/II clinical trial where CD44v6 CAR-redirected suicidal T cells will be applied soon after autologous HSCT for sterilizing residual tumor, and routinely ablated thereafter not to interfere will full hematological reconstitution. The co-expression of a suicide gene will also allow to control any potential toxicity deriving from background expression of CD44v6 on healthy tissues. Disclosures:Bordignon:Molmed SpA: Employment.

Multiple myeloma–related deregulation of bone marrow–derived CD34+ hematopoietic stem and progenitor cells

AbstractMultiple myeloma (MM) is a clonal plasma cell disorder frequently accompanied by hematopoietic impairment. We show that hematopoietic stem and progenitor cells (HSPCs), in particular megakaryocyte-erythrocyte progenitors, are diminished in the BM of MM patients. Genomic profiling of HSPC subsets revealed deregulations of signaling cascades, most notably TGFβ signaling, and pathways involved in cytoskeletal organization, migration, adhesion, and cell-cycle regulation in the patients. Functionally, proliferation, colony formation, and long-term self-renewal were impaired as a consequence of activated TGFβ signaling. In accordance, TGFβ levels in the BM extracellular fluid were elevated and mesenchymal stromal cells (MSCs) had a reduced capacity to support long-term hematopoiesis of HSPCs that completely recovered on blockade of TGFβ signaling. Furthermore, we found defective actin assembly and down-regulation of the adhesion receptor CD44 in MM HSPCs functionally reflected by impaired migration and adhesion. Still, transplantation into myeloma-free NOG mice revealed even enhanced engraftment and normal differentiation capacities of MM HSPCs, which underlines that functional impairment of HSPCs depends on MM-related microenvironmental cues and is reversible. Taken together, these data implicate that hematopoietic suppression in MM emerges from the HSPCs as a result of MM-related microenvironmental alterations.

Heterogeneous nuclear ribonucleoprotein L-like (hnRNPLL) and elongation factor, RNA polymerase II, 2 (ELL2) are regulators of mRNA processing in plasma cells

B cells and plasma cells possess distinct RNA processing environments that respectively promote the expression of membrane-associated Ig by B cells versus the secretion of Ig by plasma cells. Through a combination of transcriptional profiling and screening using a lentiviral short-hairpin RNA interference library, we show that both the splicing factor hnRNPLL and the transcription elongation factor ELL2 modulate the ratio of secreted versus membrane-encoding Ighg2b transcripts in MPC11 plasmacytoma cell lines. hnRNPLL and ELL2 are both highly expressed in primary plasma cells relative to B cells, but hnRNPLL binds Ighg2b mRNA transcripts and promotes an increase in levels of the membrane-encoding Ighg2b isoform at the expense of the secreted Ighg2b isoform, whereas ELL2 counteracts this effect and drives Ig secretion by increasing the frequency of the secreted Ighg2b isoform. As in T cells, hnRNPLL also alters the splicing pattern of mRNA encoding the adhesion receptor CD44, promoting exon inclusion, and decreasing the overall level of CD44 expression. Further characterization of ELL2-dependent transcription by RNA-Seq revealed that ∼12% of transcripts expressed by plasma cells were differentially processed because of the activities of ELL2, including B-cell maturation antigen BCMA, a receptor with a defined role in plasma cell survival. Taken together, our data identify hnRNPLL and ELL2 as regulators of pre-mRNA processing in plasma cells.

Dynamic actin remodeling during epithelial–mesenchymal transition depends on increased moesin expression

Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.

CD44 Promotes Epithelial Mammary Gland Development and Exhibits Altered Localization during Cancer Progression

The basal cell layer has emerged as a critical player in cancer progression, and understanding the molecular contribution of specific cell types is important in treatment and prevention. The adhesion receptor CD44, which mediates epithelial-stromal and cell-cell interactions, has been shown to both promote and suppress tumor progression. To better understand the normal function of CD44, we have investigated its role in mouse mammary gland development and its expression in human breast and prostate cancer. We have found that CD44 is expressed in the myoepithelium of the developing mammary gland and modulates ductal development of FVB/N mice. The loss of CD44 results in defective luminal-myoepithelial cell-cell adhesion and promotes the mixing of luminal and myoepithelial layers, disrupting epithelial bilayer organization, and CD44-null mice experience delayed ductal outgrowth and impaired terminal end bud formation. The myoepithelial expression of CD44 is also relevant to its expression in cancer, as CD44 is expressed in the basal cells of early-stage breast and prostate cancer but exhibits altered localization with increasing tumorigenicity and is strongly expressed by tumor epithelium.

Abstract 3628: The forced interaction of α4 integrin and CD44 by the novel peptide HYD1 may be a determinant of HYD1-induced cell death in multiple myeloma

Abstract Our laboratory has previously shown that treatment with the novel d-amino acid peptide HYD1 (kikmviswkg) induces necrotic cell death in multiple myeloma (MM) cell lines. Furthermore, we have implicated the adhesion receptor VLA-4 as being involved in this process via shRNA silencing strategies. However, reducing α4 integrin only demonstrated partial protection indicating that other components of the HYD1 binding complex may be required for cell death. Thus, in the present study, we investigated the possible role of other adhesion molecules and/or complexes that may contribute to HYD1-induced cell death. Using an unbiased approach, we performed a total membrane pull-down with biotin conjugated HYD1 and NeutrAvidin beads on NCI-H929 MM cells. Samples were then loaded on an SDS-PAGE gel and analyzed by Mass Spectrometry. Data were then mined with the Scaffold 3 proteome software to determine pertinent hits. The adhesion receptor CD44 was determined to have the most peptide match hits and was confirmed as a valid hit via Western Blot analysis. Preliminary data using ELISA based binding studies with biotin-HYD1 as bait shows that recombinant CD44 directly binds to biotin-HYD1 in a concentration dependent manner. In order to identify complexes involved in HYD1-induced cell death, membrane immunoprecipitation assays were performed using CD44 and α4 integrin mAbs in both the presence and absence of HYD1. While Western Blot analysis confirmed basal levels of complex interactions between α4 integrin and CD44, the complex was significantly enhanced in the presence of HYD1. These data not only indicate a possible role of CD44 in HYD1-induced cell death, but also the ability of HYD1 to induce interactions among adhesion molecules and complexes. Further studies, including CD44shRNA targeting strategies, as well as looking at other adhesion molecules will be needed in order to confirm the role of individual cell surface receptors identified in the biotin-HYD1 complex in mediating HYD1 induced cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3628. doi:10.1158/1538-7445.AM2011-3628

Go or Grow: How HA Controls the EGF Receptor

The extracellular matrix (ECM) can govern epithelial cell behavior by influencing cellular invasion, a process which signals a deadly stage of cancer progression. In attempting to understand the role of the ECM in invasion, we previously found that the adhesion receptor CD44 protects against metastasis when cells are presented with hyaluronan (HA) embedded in Type I collagen. In our current studies, we have attempted to determine the mechanism by which CD44/HA interactions are inhibiting invasion. We have discovered that the inhibition of invasion provided by collagen‐embedded HA (eHA) occurs through the down‐regulation of both the Epidermal Growth Factor Receptor (EGFR) and PAK and corresponds with cell‐cell attachment and lamellopodia formation. Of significance is the observation that when HA is provided to cells in a soluble form (sHA), invasion is promoted and EGFR and PAK are both activated, corresponding to an inhibition of cell‐cell attachment and filopodia formation. Finally, we have found that these opposing effects of eHA versus sHA are further carried to the arena of proliferation, as eHA promotes proliferation and ERK MAP Kinase activity, while sHA inhibits each of these activities in the presence of collagen. Together, these data demonstrate that the form of HA presentation to the cell has a dramatic affect on its ability to alter cellular morphology, behavior and signal transduction events.

Inhibition of Platelet-derived Growth Factor-BB-induced Receptor Activation and Fibroblast Migration by Hyaluronan Activation of CD44

The extracellular matrix molecule hyaluronan was found to suppress platelet-derived growth factor (PDGF) beta-receptor activation and PDGF-BB-induced migration of primary human dermal fibroblasts. The suppressive effect of hyaluronan was neutralized by a monoclonal antibody that specifically inhibits hyaluronan binding to its receptor CD44. Moreover, co-immunoprecipitation experiments showed that the PDGF beta-receptor and CD44 can form a complex. Interestingly, the inhibitory effect of hyaluronan on PDGF beta-receptor activation was not seen in the presence of the tyrosine phosphatase inhibitor pervanadate. Our observations suggest that hyaluronan suppresses PDGF beta-receptor activation by recruiting a CD44-associated tyrosine phosphatase to the receptor.

Perioperative cyclooxygenase 2 inhibition to reduce tumor cell adhesion and metastatic potential of circulating tumor cells in non–small cell lung cancer

Surgical manipulation of lung cancers may increase circulating tumor cells and contribute to metastatic recurrence after resection. Cyclooxygenase 2 is overexpressed in most non-small cell lung cancer and upregulates the cell adhesion receptor CD44. Our goal was to examine the effects of perioperative cyclooxygenase blockade on the metastatic potential of circulating tumor cells, CD44 expression, and adhesion of cancer cells to extracellular matrix. Human non-small cell lung cancer cells (A549) were injected through the lateral tail vein in an in vivo murine model of tumor metastasis with three random treatment groups: no treatment, perioperative selective cyclooxygenase 2 inhibition (celecoxib) only, and continuous celecoxib. Lung metastases were assessed at 6 weeks by a blinded observer. For in vitro experiments, cells were treated with celecoxib, and expression of CD44 was determined by Western blotting. Extracellular matrix adhesion was assessed by Matrigel (BD Labware, Bedford, Mass) assay. In vivo lung metastases were significantly decreased relative to control by both perioperative and continuous celecoxib (P = .0135). There was no significant difference in number of metastases between continuous and perioperative treatment groups. In vitro adhesion to the extracellular matrix was significantly inhibited by celecoxib in a dose-dependent manner (P < .01). A549 cells expressed high levels of CD44, upregulated by interleukin 1beta and downregulated by celecoxib. Celecoxib significantly reduced establishment of metastases by circulating tumor cells in a murine model. It also inhibited CD44 expression and extracellular matrix adhesion in vitro. Perioperative modulation of cyclooxygenase 2 may be a novel strategy to minimize metastases from circulating tumor cells during this high-risk period.

CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+stem/progenitor cells to bone marrow

AbstractTrafficking of human CD34+stem/progenitor cells (HSCs/HPCs) is regulated by chemokines, cytokines, proteolytic enzymes, and adhesion molecules. We report that the adhesion receptor CD44 and its major ligand, hyaluronic acid (HA), are essential for homing into the bone marrow (BM) and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and engraftment by human HSCs. Homing was blocked by anti-CD44 monoclonal antibodies (mAbs) or by soluble HA, and it was significantly impaired after intravenous injection of hyaluronidase. Furthermore, stromal cell-derived factor-1 (SDF-1) was found to be a rapid and potent stimulator of progenitor adhesion to immobilized HA, leading to formation of actin-containing protrusions with CD44 located at their tips. HPCs migrating on HA toward a gradient of SDF-1 acquired spread and polarized morphology with CD44 concentrating at the pseudopodia at the leading edge. These morphologic alterations were not observed when the progenitors were first exposed to anti-CD44 mAbs, demonstrating a crosstalk between CD44 and CXCR4 signaling. Unexpectedly, we found that HA is expressed on human BM sinusoidal endothelium and endosteum, the regions where SDF-1 is also abundant. Taken together, our data suggest a key role for CD44 and HA in SDF-1–dependent transendothelial migration of HSCs/HPCs and their final anchorage within specific niches of the BM. (Blood. 2004;103:2981-2989)

A new spreadsheet method for the analysis of bivariate flow cytometric data

BackgroundA useful application of flow cytometry is the investigation of cell receptor-ligand interactions. However such analyses are often compromised due to problems interpreting changes in ligand binding where the receptor expression is not constant. Commonly, problems are encountered due to cell treatments resulting in altered receptor expression levels, or when cell lines expressing a transfected receptor with variable expression are being compared. To overcome this limitation we have developed a Microsoft Excel spreadsheet that aims to automatically and effectively simplify flow cytometric data and perform statistical tests in order to provide a clearer graphical representation of results.ResultsTo demonstrate the use and advantages of this new spreadsheet method we have investigated the binding of the transmembrane adhesion receptor CD44 to its ligand hyaluronan. In the first example, phorbol ester treatment of cells results in both increased CD44 expression and increased hyaluronan binding. By applying the spreadsheet method we effectively demonstrate that this increased ligand binding results from receptor activation. In the second example we have compared AKR1 cells transfected either with wild type CD44 (WT CD44) or a mutant with a truncated cytoplasmic domain (CD44-T). These two populations do not have equivalent receptor expression levels but by using the spreadsheet method hyaluronan binding could be compared without the need to generate single cell clones or FACS sorting the cells for matching CD44 expression. By this method it was demonstrated that hyaluronan binding requires a threshold expression of CD44 and that this threshold is higher for CD44-T. However, at high CD44-T expression, binding was equivalent to WT CD44 indicating that the cytoplasmic domain has a role in presenting the receptor at the cell surface in a form required for efficient hyaluronan binding rather than modulating receptor activity.ConclusionUsing the attached spreadsheets and instructions, a simple post-acquisition method for analysing bivariate flow cytometry data is provided. This method constitutes a straightforward improvement over the standard graphical output of flow cytometric data and has the significant advantage that ligand binding can be compared between cell populations irrespective of receptor expression levels.