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1.  Abrogating Drug Resistance in Malignant Peripheral Nerve Sheath Tumors by Disrupting Hyaluronan-CD44 Interactions with Small Hyaluronan Oligosaccharides 
Cancer research  2009;69(12):4992-4998.
Malignant peripheral nerve sheath tumors (MPNST) develop in ~10% of neurofibromatosis type-1 patients and are a major contributing factor to neurofibromatosis-1 patient mortality and morbidity. MPNSTs are multidrug resistant, and thus long-term patient survival rates are poor after standard doxorubicin or multiagent chemotherapies. We show that the hyaluronan receptor CD44 forms complexes with multidrug transporters, BCRP (ABCG2) and P-glycoprotein (ABCB1), in the plasma membrane of human MPNST cells. Small hyaluronan oligosaccharides antagonize hyaluronan-CD44–mediated processes and inhibit hyaluronan production. Treatment of MPNST cells with the hyaluronan oligomers causes disassembly of CD44-transporter complexes and induces internalization of CD44, BCRP, and P-glycoprotein. Consequently, the oligomers suppress drug transporter activity and increase sensitivity to doxorubicin treatment in culture. In vivo, systemic administration of hyaluronan oligomers inhibits growth of MPNST xenografts. Moreover, the oligomers and doxorubicin act synergistically in vivo, in that combined suboptimal doses induce tumor regression to a greater extent than the additive effects of each agent alone. These findings indicate that constitutive hyaluronan-CD44 interactions contribute to drug transporter localization and function at the plasma membrane, and that attenuating hyaluronan-CD44 interactions sensitizes MPNSTs to doxorubicin in vitro and in vivo. These results also show the potential efficacy of hyaluronan oligomers, which are nontoxic and nonimmunogenic, as an adjuvant for chemotherapy in MPNST patients.
PMCID: PMC3655760  PMID: 19470767
2.  Inhibition of functional hyaluronan-CD44 interactions in CD133-positive primary human ovarian carcinoma cells by small hyaluronan oligosaccharides 
CD44 is one of the most common markers used for identification of highly tumorigenic sub-populations of human carcinoma cells, but little is known about the function of CD44 or its major ligand, hyaluronan, in these cells. The purpose of this study is to investigate the involvement of hyaluronan and its interaction with CD44 in the properties of a tumorigenic sub-population of primary ovarian carcinoma cells.
Experimental Design
A tumorigenic sub-population was identified in ascites fluids from ovarian carcinoma patients by expression of high CD133 levels. Treatment with small hyaluronan oligosaccharides, which dissociate constitutive hyaluronan polymer-CD44 interactions, was used to test the importance of hyaluronan-CD44 interaction in assembly of multidrug and monocarboxylate transporters and receptor tyrosine kinases in the plasma membrane of cells with high CD133 levels, and in the tumorigenic capacity of the CD133-high sub-population.
Although total CD44 levels were similar in cells with high or low CD133 expression, CD44 was present in close association with transporters, receptor tyrosine kinases and emmprin (CD147) in the plasma membrane of cells with high CD133 levels. Treatment with small hyaluronan oligosaccharides reduced association of the transporters and receptor tyrosine kinases with CD44 in the plasma membrane, diminished drug transporter activity, and inhibited intra-peritoneal tumorigenesis in these cells.
We conclude that hyaluronan-CD44 interaction plays an important role in the properties of highly tumorigenic cells by stabilizing oncogenic complexes in their plasma membrane, and that treatment with hyaluronan-CD44 antagonists provides a logical therapeutic approach for abrogating the properties of these cells.
PMCID: PMC2794991  PMID: 19996211
3.  Hyaluronan, CD44, and Emmprin Regulate Lactate Efflux and Membrane Localization of Monocarboxylate Transporters in Human Breast Carcinoma Cells 
Cancer research  2009;69(4):1293-1301.
Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin)is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells.
PMCID: PMC3655770  PMID: 19176383
4.  Direct activation of emmprin and associated pathogenesis by an oncogenic herpesvirus 
Cancer research  2010;70(10):3884-3889.
Emmprin is a multifunctional glycoprotein expressed by cancer cells and stromal cells in the tumor microenvironment. Through both direct effects within tumor cells and promotion of tumor-stroma interactions, emmprin induces tumor cell invasiveness and regional angiogenesis. The Kaposi’s sarcoma-associated herpesvirus (KSHV) is a common etiology of cancers arising in the setting of immune suppression, including Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL). However, whether emmprin expression and function are regulated by KSHV or other oncogenic viruses in the tumor microenvironment to promote viral cancer pathogenesis remains unknown. Fibroblasts and endothelial cells support latent KSHV infection and represent cellular components of KS lesions. Therefore, we utilized primary human fibroblasts and endothelial cells to determine whether KSHV itself regulates emmprin expression, and whether KSHV-emmprin interactions mediate cell invasiveness. We found that KSHV promotes fibroblast and endothelial cell invasiveness following de novo infection through the upregulation of emmprin, and that this effect is mediated by the KSHV-encoded latency-associated nuclear antigen (LANA). We further validated these findings through our observations that emmprin promotes invasiveness, as well as colony formation, by PEL cells derived from human tumors. Collectively, these data implicate KSHV activation of emmprin as an important mechanism for cancer progression and support the potential utility of targeting emmprin as a novel therapeutic approach for KSHV-associated tumors.
PMCID: PMC3202426  PMID: 20406987
KSHV; CD147; Kaposi’s sarcoma; lymphoma
5.  High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2 
The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-IR). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1–6; 22–31 kDa) that via high affinity binding to the IGFs (KD ~ 300–700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in E. coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP.
PMCID: PMC2934857  PMID: 20541521
Recombinant human IGF- binding protein-2 (rhIGFBP-2); E coli Expression; Protein Purification; Protein Structure; Secondary Structure; G-matrix Fourier transform (GFT) NMR
6.  Regulation of Invasive Behavior by Vascular Endothelial Growth Factor is HEF1-dependent 
Oncogene  2010;29(31):4449-4459.
We previously reported a VEGF autocrine loop in head and neck squamous cell carcinoma (HNSCC) cell lines, supporting a role for VEGF in HNSCC tumorigenesis. Using a phosphotyrosine proteomics approach we screened the HNSCC cell line, SCC-9 for effectors of VEGFR2 signaling. A cluster of proteins involved in cell migration and invasion, including the p130Cas paralog, human enhancer of filamentation1 (HEF1/Cas-L/Nedd9) was identified. HEF1 silencing and overexpression studies revealed a role for VEGF in regulating cell migration, invasion, and MMP expression in a HEF1-dependent manner. Moreover, cells plated on extracellular matrix coated coverslips exhibited enhanced invadopodia formation in response to VEGF that was HEF1-dependent. Immunolocalization revealed that HEF1 colocalized to invadopodia with MT1-MMP. Analysis of HNSCC tissue microarrays for HEF1 immunoreactivity revealed a 6.5-fold increase in the odds of having a metastasis with a high HEF1 score compared to a low HEF1 score. These findings suggest that HEF1 may be prognostic for advanced stage HNSCC. They also demonstrate for the first time, that HEF1 is required for VEGF-mediated HNSCC cell migration and invasion, consistent with HEF1’s recent identification as a metastatic regulator. These results support a strategy targeting VEGF:VEGFR2 in HNSCC therapeutics.
PMCID: PMC2921319  PMID: 20498643
VEGF; Invadopodia; Cell migration; invasion; HEF1; NEDD9; CAS-L; tyrosine phosphorylation
7.  Hyaluronan: a constitutive regulator of chemoresistance and malignancy in cancer cells 
Seminars in cancer biology  2008;18(4):244-250.
Hyaluronan is an important structural component of extracellular matrices but also interacts instructively with cells during embryonic development, healing processes, inflammation, and cancer. It binds to several different types of cell surface receptors, including CD44, thus leading to co-regulation of important signaling pathways, notably those induced by activation of receptor tyrosine kinases. Consequently, interactions of both stromal and tumor cell-derived hyaluronan with tumor cells play important cooperative roles in several aspects of malignancy. This review focuses on cell autonomous hyaluronan-tumor cell interactions that lead to activation of receptor tyrosine kinases and enhanced drug resistance. Particular emphasis is placed on the role of hyaluronan-CD44 interactions in drug transporter expression and activity, especially in cancer stem-like cells that are highly malignant and resistant to chemotherapy. Antagonists of hyaluronan-CD44 interaction, especially small hyaluronan oligomers, may be useful in therapeutic strategies aimed at preventing tumor recurrence from these therapy-resistant sub-populations within malignant cancers.
PMCID: PMC2517221  PMID: 18534864
8.  Hyaluronan, CD44 and Emmprin: Partners in cancer cell chemoresistance 
Hyaluronan is not only an important structural component of extracellular matrices but also interacts with cells during dynamic cell processes such as occur in cancer. Consequently, interactions of hyaluronan with tumor cells play important cooperative roles in various aspects of malignancy. Hyaluronan binds to several cell surface receptors, including CD44, thus leading to co-regulation of signaling pathways that are important in regulation of multidrug resistance to anticancer drugs, in particular anti-apoptotic pathways induced by activation of receptor tyrosine kinases. Emmprin, a cell surface glycoprotein of the Ig superfamily, stimulates hyaluronan production and downstream signaling consequences. Emmprin and CD44 also interact with various multidrug transporters of the ABC family and monocarboxylate transporters associated with resistance to cancer therapies. Moreover, hyaluronan-CD44 interactions are critical to these properties in the highly malignant, chemotherapy-resistant cancer stem-like cells. Perturbations of the hyaluronan-CD44 interaction at the plasma membrane by various antagonists result in attenuation of receptor tyrosine kinase and transporter activities and inhibition of tumor progression in vivo. These antagonists, especially small hyaluronan oligomers, may be useful in therapeutic strategies aimed at preventing tumor refractoriness or recurrence due to drug-resistant sub-populations within malignant cancers.
PMCID: PMC2584579  PMID: 18490190
hyaluronan; CD44; emmprin; CD147; cancer stem cells; multidrug resistance; receptor tyrosine kinases; ABC-family drug transporters; monocarboxylate transporters

Results 1-8 (8)