IGF-dependent dynamic modulation of a protease cleavage site in the intrinsically disordered linker domain of human IGFBP2.

Functional regulation via conformational dynamics is well known in structured proteins but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy, we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 (hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L-hIGFBP2) retains its intrinsic disorder upon binding IGF-1, but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.

CD147-dependent heterogeneity in malignant and chemoresistant properties of cancer cells.

CD147 (alias emmprin or basigin), an integral plasma membrane glycoprotein and a member of the Ig superfamily, is widespread in normal tissues, but highly up-regulated in many types of malignant cancer cells. CD147 is multifunctional, with numerous binding partners. Recent studies suggest that complexes of CD147 with the hyaluronan receptor CD44 and associated transporters and receptor tyrosine kinases are enriched in the plasma membrane of cancer stem-like cells. Here, we show that subpopulations of tumor cell lines constitutively expressing high levels of cell-surface CD147 exhibit cancer stem-like cell properties; that is, they exhibit much greater invasiveness, anchorage-independent growth, spheroid formation, and drug resistance in vitro and higher tumorigenicity in vivo than those constitutively expressing low levels of cell-surface CD147. Primary CD147-rich cell subpopulations derived from mouse mammary adenocarcinomas also exhibit high levels of invasiveness and spheroid-forming capacity, whereas CD147-low cells do not. Moreover, localization at the plasma membrane of CD44, the EGF receptor, the ABCB1 and ABCG2 drug transporters, and the MCT4 monocarboxylate transporter is elevated in cells constitutively expressing high levels of cell-surface CD147. These results show that CD147 is associated with assembly of numerous pro-oncogenic proteins in the plasma membrane and may play a fundamental role in properties characteristic of cancer stem-like cells.

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-1R). 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 (K(D) approximately 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 Escherichia 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.

Direct activation of emmprin and associated pathogenesis by an oncogenic herpesvirus.

Emmprin (extracellular matrix metalloproteinase inducer) 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 for cancers arising in the setting of immune suppression, including Kaposi's sarcoma and primary effusion lymphoma. 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 Kaposi's sarcoma lesions. Therefore, we used 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. We also found that emmprin promotes invasiveness, as well as colony formation, by primary effusion lymphoma 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.

Inhibition of Functional Hyaluronan-CD44 Interactions in CD133-positive Primary Human Ovarian Carcinoma Cells by Small Hyaluronan Oligosaccharides.

PURPOSE: CD44 is one of the most common markers used for identification of highly tumorigenic subpopulations 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 was to investigate the involvement of hyaluronan and its interaction with CD44 in the properties of a tumorigenic subpopulation of primary ovarian carcinoma cells. EXPERIMENTAL DESIGN: A tumorigenic subpopulation 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 subpopulation. RESULTS: 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 i.p. tumorigenesis in these cells. CONCLUSIONS: 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. (Clin Cancer Res 2009;15(24):7593-601).

Abrogating drug resistance in malignant peripheral nerve sheath tumors by disrupting hyaluronan-CD44 interactions with small hyaluronan oligosaccharides.

Malignant peripheral nerve sheath tumors (MPNST) develop in approximately 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.

Hyaluronan, CD44, and emmprin regulate lactate efflux and membrane localization of monocarboxylate transporters in human breast carcinoma cells.

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.

Hyaluronan: a constitutive regulator of chemoresistance and malignancy in cancer cells.

Hyaluronan not only 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.

Hyaluronan, CD44 and Emmprin: partners in cancer cell chemoresistance.

Hyaluronan not only is an important structural component of extracellular matrices but also interacts with cells during dynamic cell processes such as those occurring 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.

Insulin-like growth factor-1 receptor and ligand targeting in head and neck squamous cell carcinoma.

IGF-1 receptor (IGF-1R) signaling is associated with increased tumorigenesis of epithelial cancers. In light of recent epidemiological studies correlating high circulating levels of IGF-1 with increased risk of second primary tumors (SPTs) of the head and neck, we examined IGF system and epidermal growth factor receptor (EGFR) expression in human head and neck squamous cell carcinoma (HNSCC) matched pairs and a cross-section of HNSCC cell lines. Employing the latter, we demonstrated that IGF-1 stimulated S-phase transition in a PI 3-K/Akt and Erk-dependent manner in 5 of 8 cell lines, with Erk activation being dependent upon EGFR kinase activity. IGF-1 stimulated thymidine incorporation was inhibited by treatment with IGFBP-3, the IGF-1R tyrosine kinase inhibitor NVP-AEW541, or the EGFR tyrosine kinase inhibitor AG1478. Combining IGFBP-3 with NVP-AEW541 or AG1478 abrogated IGF-1 responses at 10-fold lower doses than either compound alone. These results demonstrate the potential for co-targeting the IGF system and EGFR in HNSCC.

Hypoxia-inducible factor-1-dependent and -independent regulation of insulin-like growth factor-1-stimulated vascular endothelial growth factor secretion.

Hypoxia-induced stress plays a central role in retinal vascular disease and cancer. Increased hypoxia-inducible factor-1 alpha (Hif-1 alpha) expression leads to HIF-1 formation and the production of vascular endothelial growth factor (VEGF). Cytokines, including insulin-like growth factor-1 (IGF-1), also stimulate VEGF secretion. In this study, we examined the relationship between IGF-1 signaling, HIF-1 alpha protein turnover and VEGF secretion in the ARPE-19 retinal pigment epithelial cell line. Northern analysis revealed that IGF-1 stimulated Hif-1 alpha message expression, whereas the hypoxia-mimetic CoCl2 did not. CoCl2 treatment increased Hif-1 alpha protein accumulation to a greater extent than IGF-1 treatment. However, IGF-1 stimulated a more significant increase in VEGF secretion. IGF-1-stimulated VEGF promoter activity was phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin)-dependent, whereas VEGF secretion was only partially reduced by inhibition of PI3K/Akt/mTOR and HIF-1 activities. Analysis of VEGF promoter truncation mutants indicated that sensitivity to CoCl2 was hypoxia response element (HRE)-dependent with the region upstream of the HRE conferring IGF-1 sensitivity. In conclusion, IGF-1 regulates VEGF expression and secretion via HIF-1-dependent and -independent pathways.

IGF-1 induced vascular endothelial growth factor secretion in head and neck squamous cell carcinoma.

Elevated vascular endothelial growth factor (VEGF) levels correlate with increased progression and poor prognosis of head and neck squamous cell carcinomas (HNSCC). VEGF expression is regulated by hypoxia and cytokines, including insulin-like growth factor-1 (IGF-1). In this report, we examined IGF-1 signaling and VEGF expression in SCC-9 cells. IGF-1 and the chemical hypoxia agent, cobalt chloride, each stimulated VEGF secretion and VEGF promoter activation. Cobalt chloride increased Hif-1alpha protein levels and HIF-1 dependent activation of the enolase promoter. IGF-1 increased these parameters only in the presence of cobalt chloride. IGF-1 stimulated PI-3K/Akt and Erk/MAPK pathways in SCC-9 cells, each contributing to Hif-1alpha expression and VEGF secretion. SCC-9 cells express the VEGF receptors Flk-1 and neuropilin-1, with VEGF treatment increasing VEGF promoter activity and VEGF secretion that was attenuated by the Flk-1 tyrosine kinase inhibitor, ZM 323881. These results demonstrate the presence of an IGF-1 regulated VEGF autocrine loop in HNSCC.

IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407.

PURPOSE: To examine insulin-like growth factor (IGF)-1 stimulation of expression of hypoxia inducible factor (HIF)-1 alpha and secretion of vascular endothelial growth factor (VEGF) and IGF binding protein (IGFBP)-3 in human retinal pigment epithelial (RPE) cell line D407. METHODS: D407 cells cultured in dishes or Transwell inserts were treated with cobalt chloride or varying doses of IGF-1. Whole cell lysates were assayed by immunoblot for HIF-1 alpha expression, whereas conditioned medium was TCA precipitated and assayed by immunoblot for VEGF and ligand blot for IGFBP-3. Cells grown on coverslips were similarly treated and probed with antibodies to HIF-1 alpha, VEGF, and IGFBP-3, and visualized by epifluorescence microscopy. Cells grown on Transwell inserts were probed with antibodies to the Na(+)/K(+)-ATPase alpha-1 subunit and either the alpha or beta subunits of the IGF-1 receptor and visualized in Z-section using confocal microscopy. RESULTS: Immunoblot analysis of whole cell lysates from IGF-1-treated D407 cells revealed the upregulation of HIF-1 alpha protein. Epifluorescence microscopy demonstrated a positive correlation between HIF-1 alpha expression and nuclear localization, VEGF and IGFBP-3 synthesis and export, and IGF-1 action. Western and ligand blot analyses of RPE cell-conditioned medium indicated that IGF-1 induced increases in VEGF and IGFBP-3 secretion. Cells grown on Transwell inserts exhibited constitutive apical secretion of VEGF and IGFBP-3, which increased on apical or basolateral treatment with IGF-1. Confocal analysis of Transwell-cultured D407 cells confirmed the apical localization of the Na(+)/K(+)-ATPase alpha-1 subunit, characteristic of polarized RPE, with IGF-1 receptor alpha and beta subunits exhibiting a nonpolarized distribution. CONCLUSIONS: IGF-1 stimulates increased HIF-1 alpha expression as well as VEGF and IGFBP-3 secretion in D407 cells. Similar to their in vivo counterparts, D407 cells maintain reversed epithelial polarity. Apical secretion of VEGF and IGFBP-3 increases in response to either apical or basolateral IGF-1 stimulation consistent with the nonpolarized distribution of IGF-1 receptors.

Autocrine effects of IGF-I-induced VEGF and IGFBP-3 secretion in retinal pigment epithelial cell line ARPE-19.

Hypoxia-induced physiological stress plays a central role in various neovascular diseases of the eye. Increased expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and subsequent formation of HIF-1 dimers active at the vascular endothelial growth factor (VEGF) promoter lead to expression of this potent angiogenic factor in the retina, including retinal pigment epithelial (RPE) cells. We previously demonstrated that insulin-like growth factor I (IGF-I) stimulates VEGF and IGF binding protein (IGFBP)-3 secretion in RPE cells. In this study we examined IGF-I-induced HIF-1alpha expression, VEGF and IGFBP-3 secretion, and the autocrine actions of VEGF and IGFBP-3 on these processes in the spontaneously transformed RPE cell line ARPE-19. Cells were treated with CoCl(2), IGF-I, recombinant human (rh)IGFBP-3, and rhVEGF. Immunoblot analysis revealed IGF-I-induced upregulation of total HIF-1alpha protein, whereas luciferase reporter assays of HIF-1 transcriptional activity demonstrated accumulation of HIF-1alpha correlated with the formation of functional HIF-1 heterodimers. Western and ligand blot analyses of RPE cell conditioned medium confirmed that IGF-I stimulated VEGF and IGFBP-3 secretion. rhVEGF stimulated IGFBP-3 secretion in an IGF-I- and HIF-1alpha-independent manner, whereas rhIGFBP-3 attenuated IGF-I-induced VEGF secretion. These findings demonstrate the multifaceted autocrine regulation of IGF-I-induced VEGF secretion by IGFBP-3 secreted in response to both IGF-I and, to a lesser extent, VEGF. These results provide evidence for HIF-1-dependent and -independent mechanisms by which IGF-I regulates VEGF and IGFBP-3 secretion.