Lu Dai

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.

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 Kaposis sarcoma-associated herpesvirus (KSHV) is a common etiology for cancers arising in the setting of immune suppression, including Kaposis 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 Kaposis 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.

Targeting sphingosine kinase induces apoptosis and tumor regression for KSHV-associated primary effusion lymphoma

Sphingosine kinase (SPHK) is overexpressed by a variety of cancers, and its phosphorylation of sphingosine results in accumulation of sphingosine-1-phosphate (S1P) and activation of antiapoptotic signal transduction. Existing data indicate a role for S1P in viral pathogenesis, but roles for SPHK and S1P in virus-associated cancer progression have not been defined. Rare pathologic variants of diffuse large B-cell lymphoma arise preferentially in the setting of HIV infection, including primary effusion lymphoma (PEL), a highly mortal tumor etiologically linked to the Kaposis sarcoma-associated herpesvirus (KSHV). We have found that ABC294640, a novel clinical-grade small molecule selectively targeting SPHK (SPHK2 >> SPHK1), induces dose-dependent caspase cleavage and apoptosis for KSHV+ patient-derived PEL cells, in part through inhibition of constitutive signal transduction associated with PEL cell proliferation and survival. These results were validated with induction of PEL cell apoptosis using SPHK2-specific siRNA, as well as confirmation of drug-induced SPHK inhibition in PEL cells with dose-dependent accumulation of proapoptotic ceramides and reduction of intracellular S1P. Furthermore, we demonstrate that systemic administration of ABC294640 induces tumor regression in an established human PEL xenograft model. Complimentary ex vivo analyses revealed suppression of signal transduction and increased KSHV lytic gene expression within drug-treated tumors, with the latter validated in vitro through demonstration of dose-dependent viral lytic gene expression within PEL cells exposed to ABC294640. Collectively, these results implicate interrelated mechanisms and SPHK2 inhibition in the induction of PEL cell death by ABC294640 and rationalize evaluation of ABC294640 in clinical trials for the treatment of KSHV-associated lymphoma. Mol Cancer Ther; 13(1); 154–64. ©2013 AACR.

Targeting xCT, a cystine-glutamate transporter induces apoptosis and tumor regression for KSHV/HIV-associated lymphoma.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of primary effusion lymphoma (PEL), which represents a rapidly progressing malignancy arising in HIV-infected patients. Conventional chemotherapy for PEL treatment induces unwanted toxicity and is ineffective — PEL continues to portend nearly 100% mortality within a period of months, which requires novel therapeutic strategies. The amino acid transporter, xCT, is essential for the uptake of cystine required for intracellular glutathione (GSH) synthesis and for maintaining the intracellular redox balance. Inhibition of xCT induces growth arrest in a variety of cancer cells, although its role in virus-associated malignancies including PEL remains unclear. In the current study, we identify that xCT is expressed on the surface of patient-derived KSHV+ PEL cells, and targeting xCT induces caspase-dependent cell apoptosis. Further experiments demonstrate the underlying mechanisms including host and viral factors: reducing intracellular GSH while increasing reactive oxygen species (ROS), repressing cell-proliferation-related signaling, and inducing viral lytic genes. Using an immune-deficient xenograft model, we demonstrate that an xCT selective inhibitor, Sulfasalazine (SASP), prevents PEL tumor progression in vivo. Together, our data provide innovative and mechanistic insights into the role of xCT in PEL pathogenesis, and the framework for xCT-focused therapies for AIDS-related lymphoma in future.

Staphylococcus epidermidis recovered from indwelling catheters exhibit enhanced biofilm dispersal and "self-renewal" through downregulation of agr.

BackgroundIn recent years, Staphylococcus epidermidis ( Se) has become a major nosocomial pathogen and the most common cause of infections of implanted prostheses and other indwelling devices. This is due in part to avid biofilm formation by Se on device surfaces. However, it still remains unknown that how the process of Se biofilm development is associated with relapsed infection in such patients.ResultsWe have identified clinical Se isolates displaying enhanced biofilm dispersal and self-renewal relative to reference strain. These isolates also exhibit enhanced initial cell attachment, extracellular DNA release, cell autolysis and thicker microcolonies during biofilm development relative to reference strain. Our genetic analyses suggest that these clinical isolates exhibit significant downregulation of RNAIII, the effector molecule of the agr quorum sensing system, and upregulation of the autolysin gene atlE. Isogenic deletion of the agr system in Se 1457 confirmed that agr negatively regulating atlE resulted in enhanced initial cell attachment, extracellular DNA release, cell autolysis and biofilm formation abilities. In contrast, double deletion of agr and atlE significantly abolished these features.ConclusionsCollectively, these data reveal the role of agr system in long-term biofilm development and pathogenesis during Se caused indwelling devices-related relapsed infection.

Lipoteichoic Acid (LTA) and Lipopolysaccharides (LPS) from Periodontal Pathogenic Bacteria Facilitate Oncogenic Herpesvirus Infection within Primary Oral Cells

Kaposi’s sarcoma (KS) remains the most common tumor arising in patients with HIV/AIDS, and involvement of the oral cavity represents one of the most common clinical manifestations of this tumor. HIV infection incurs an increased risk for periodontal diseases and oral carriage of a variety of bacteria. Whether interactions involving pathogenic bacteria and oncogenic viruses in the local environment facilitate replication or maintenance of these viruses in the oral cavity remains unknown. In the current study, our data indicate that pretreatment of primary human oral fibroblasts with two prototypical pathogen-associated molecular patterns (PAMPs) produced by oral pathogenic bacteria–lipoteichoic acid (LTA) and lipopolysaccharide (LPS), increase KSHV entry and subsequent viral latent gene expression during de novo infection. Further experiments demonstrate that the underlying mechanisms induced by LTA and/or LPS include upregulation of cellular receptor, increasing production of reactive oxygen species (ROS), and activating intracellular signaling pathways such as MAPK and NF-κB, and all of which are closely associated with KSHV entry or gene expression within oral cells. Based on these findings, we hope to provide the framework of developing novel targeted approaches for treatment and prevention of oral KSHV infection and KS development in high-risk HIV-positive patients.

Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposis sarcoma (KS), and KSHV activation of mitogen-activated protein kinases (MAPKs) initiates a number of key pathogenic determinants of KS. Direct inhibition of signal transduction as a therapeutic approach presents several challenges, and a better understanding of KSHV-induced mechanisms regulating MAPK activation may facilitate the development of new treatment or prevention strategies for KS. MAPK phosphatases, including dual-specificity phosphatase-1 (DUSP1), negatively regulate signal transduction and cytokine activation through MAPK dephosphorylation or interference with effector molecule binding to MAPKs, including the extracellular signal-regulated kinase (ERK). We found that ERK-dependent latent viral gene expression, the induction of promigratory factors, and cell invasiveness following de novo infection of primary human endothelial cells are in part dependent on KSHV suppression of DUSP1 expression during de novo infection. KSHV-encoded miR-K12-11 upregulates the expression of xCT (an amino acid transporter and KSHV fusion/entry receptor), and existing data indicate a role for xCT in the regulation of 14-3-3β, a transcriptional repressor of DUSP1. We found that miR-K12-11 induces endothelial cell secretion of promigratory factors and cell invasiveness through upregulation of xCT-dependent, 14-3-3β-mediated suppression of DUSP1. Finally, proof-of-principle experiments revealed that pharmacologic upregulation of DUSP1 inhibits the induction of promigratory factors and cell invasiveness during de novo KSHV infection. These data reveal an indirect role for miR-K12-11 in the regulation of DUSP1 and downstream pathogenesis.

Targeting HGF/c-MET induces cell cycle arrest, DNA damage and apoptosis for primary effusion lymphoma

Kaposi sarcoma-associated herpesvirus (KSHV) is a principal causative agent of primary effusion lymphoma (PEL) with a poor prognosis in immunocompromised patients. However, it still lacks effective treatment which urgently requires the identification of novel therapeutic targets for PEL. Here, we report that the hepatocyte growth factor (HGF)/c-MET pathway is highly activated by KSHV in vitro and in vivo. The selective c-MET inhibitor, PF-2341066, can induce PEL apoptosis through cell cycle arrest and DNA damage, and suppress tumor progression in a xenograft murine model. By using microarray analysis, we identify many novel genes that are potentially controlled by HGF/c-MET within PEL cells. One of the downstream candidates, ribonucleoside-diphosphate reductase subunit M2 (RRM2), also displays the promising therapeutic value for PEL treatment. Our findings provide the framework for development of HGF/c-MET-focused therapy and implementation of clinical trials for PEL patients.

Sphingosine kinase-2 maintains viral latency and survival for KSHV-infected endothelial cells.

Phosphorylation of sphingosine by sphingosine kinases (SphK1 and SphK2) generates sphingosine-1-phosphate (S1P), a bioactive sphingolipid which promotes cancer cell survival and tumor progression in vivo. We have recently reported that targeting SphK2 induces apoptosis for human primary effusion lymphoma (PEL) cell lines infected by the Kaposi’s sarcoma-associated herpesvirus (KSHV), and this occurs in part through inhibition of canonical NF-κB activation. In contrast, pharmacologic inhibition of SphK2 has minimal impact for uninfected B-cell lines or circulating human B cells from healthy donors. Therefore, we designed additional studies employing primary human endothelial cells to explore mechanisms responsible for the selective death observed for KSHV-infected cells during SphK2 targeting. Using RNA interference and a clinically relevant pharmacologic approach, we have found that targeting SphK2 induces apoptosis selectively for KSHV-infected endothelial cells through induction of viral lytic gene expression. Moreover, this effect occurs through repression of KSHV-microRNAs regulating viral latency and signal transduction, including miR-K12-1 which targets IκBα to facilitate activation of NF-κB, and ectopic expression of miR-K12-1 restores NF-κB activation and viability for KSHV-infected endothelial cells during SphK2 inhibition. These data illuminate a novel survival mechanism and potential therapeutic target for KSHV-infected endothelial cells: SphK2-associated maintenance of viral latency.