Kimberly E. Foreman

Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma–associated herpesvirus

Kaposi sarcoma is considered a neoplasm of lymphatic endothelium infected with Kaposi sarcoma–associated herpesvirus. It is characterized by the expression of lymphatic lineage–specific genes by Kaposi sarcoma tumor cells. Here we show that infection of differentiated blood vascular endothelial cells with Kaposi sarcoma–associated herpesvirus leads to their lymphatic reprogramming; induction of ∼70% of the main lymphatic lineage–specific genes, including PROX1, a master regulator of lymphatic development; and downregulation of blood vascular genes.

Targeting Notch to Target Cancer Stem Cells

The cellular heterogeneity of neoplasms has been at the center of considerable interest since the “cancer stem cell hypothesis”, originally formulated for hematologic malignancies, was extended to solid tumors. The origins of cancer “stem” cells (CSC) or tumor-initiating cells (TIC; henceforth referred to as CSCs) and the methods to identify them are hotly debated topics. Nevertheless, the existence of subpopulations of tumor cells with stem-like characteristics has significant therapeutic implications. The stem-like phenotype includes indefinite self-replication, pluripotency, and, importantly, resistance to chemotherapeutics. Thus, it is plausible that CSCs, regardless of their origin, may escape standard therapies and cause disease recurrences and/or metastasis after apparently complete remissions. Consequently, the idea of selectively targeting CSCs with novel therapeutics is gaining considerable interest. The Notch pathway is one of the most intensively studied putative therapeutic targets in CSC, and several investigational Notch inhibitors are being developed. However, successful targeting of Notch signaling in CSC will require a thorough understanding of Notch regulation and the context-dependent interactions between Notch and other therapeutically relevant pathways. Understanding these interactions will increase our ability to design rational combination regimens that are more likely to prove safe and effective. Additionally, to determine which patients are most likely to benefit from treatment with Notch-targeting therapeutics, reliable biomarkers to measure pathway activity in CSC from specific tumors will have to be identified and validated. This article summarizes the most recent developments in the field of Notch-targeted cancer therapeutics, with emphasis on CSC. Clin Cancer Res; 16(12); 3141–52. ©2010 AACR.

Rational targeting of Notch signaling in cancer

Accumulating preclinical and clinical evidence supports a pro-oncogenic function for Notch signaling in several solid tumors, particularly but not exclusively in breast cancer. Notch inhibitory agents, such as γ-secretase inhibitors, are being investigated as candidate cancer therapeutic agents. Interest in therapeutic modulation of the Notch pathway has been increased by recent reports, indicating that its role is important in controlling the fate of putative ‘breast cancer stem cells’. However, as is the case for most targeted therapies, successful targeting of Notch signaling in cancer will require a considerable refinement of our understanding of the regulation of this pathway and its effects in both normal and cancer cells. Notch signaling has bidirectional ‘cross talk’ interaction with multiple other pathways that include candidate therapeutic targets. Understanding these interactions will greatly increase our ability to design rational combination regimens. To determine which patients are most likely to benefit from treatment with Notch inhibitors, it will be necessary to develop molecular tests to accurately measure pathway activity in specific tumors. Finally, mechanism-based toxicities will have to be addressed by a careful choice of therapeutic agents, combinations and regimens. This article summarizes the current state of the field, and briefly describes opportunities and challenges for Notch-targeted therapies in oncology.

Gamma secretase inhibitor blocks Notch activation and induces apoptosis in Kaposi's sarcoma tumor cells

Kaposis sarcoma (KS) is a common neoplasm in HIV-1-infected individuals causing significant morbidity and mortality. Despite recent advances, the pathogenesis of this potentially life-threatening neoplasm remains unclear, and there is currently no cure for KS. Notch proteins are known to play a fundamental role in cell fate decisions including proliferation, differentiation, and apoptosis. It is, therefore, not surprising that Notch proteins have been implicated in tumorigenesis and appear to function as either oncogenes or tumor suppressor proteins depending on cellular context. In this report, we demonstrate elevated levels of activated Notch-1, -2, and -4 in KS tumor cells in vivo and in vitro compared to endothelial cells, the precursor of the KS cell. Notch activation was confirmed through luciferase reporter assays and localization of Hes (Hairy/Enhancer of Split)-1 and Hey (Hairy/Enhancer of Split related with YRPW)1 (primary targets of the Notch pathway) in KS cell nuclei. Studies using γ-secretase inhibitors (GSI and LY-411,575), which block Notch activation, resulted in apoptosis in primary and immortalized KS cells. Similar studies injecting GSI into established KS cell tumors on mice demonstrated growth inhibition or tumor regression that was characterized by apoptosis in treated, but not control tumors. The results indicate that KS cells overexpress activated Notch and interruption of Notch signaling inhibits KS cell growth. Thus, targeting Notch signaling may be of therapeutic value in KS patients.

PROPAGATION OF A HUMAN HERPESVIRUS FROM AIDS-ASSOCIATED KAPOSI'S SARCOMA

BACKGROUND Although unique DNA sequences related to gammaherpesviruses have been found in Kaposis sarcoma lesions, it is uncertain whether this DNA encodes a virus that is able to reproduce. METHODS We isolated and propagated a filterable agent whose DNA sequences were found to be identical to those of the Kaposis sarcoma-associated herpesvirus (KSHV). We obtained early-passage spindle cells from skin lesions of patients with the acquired immunodeficiency syndrome (AIDS) who had Kaposis sarcoma and cultured them with cells of the human embryonal-kidney epithelial-cell line 293. We characterized the virus according to its effects on cellular morphology and viral replication and its appearance on electron microscopy. RESULTS KSHV was cytotoxic to 293 cells and was detected by the polymerase chain reaction (PCR) in infected cells but not uninfected ones. Cytotoxicity and positive PCR signals were consistently maintained with viral titers of 1 million per milliliter, for about 20 serial infections of 293 cells. The viral copy number was relatively low (1 to 10 copies per cell). Viral replication was confirmed by Southern blot analysis of DNA isolated from the enriched nuclear fraction of infected cells and by a semiquantitative PCR using dilutions of the lysates of infected cells to detect the 233-bp viral DNA fragment originally described in association with Kaposis lesions. Electron microscopy revealed herpesvirus-like particles in about 1 percent of cells from infected cultures, as compared with none in cells from uninfected cultures. CONCLUSIONS A herpesvirus with DNA sequences identical to those of KSHV can be propagated from skin lesions of patients with AIDS-associated Kaposis sarcoma.

Induction of HHV-8 Lytic Cycle Replication by Inflammatory Cytokines Produced by HIV-1-Infected T Cells

Human herpesvirus 8 (HHV-8) is a gamma2-herpesvirus consistently identified in Kaposis sarcoma (KS), primary effusion lymphoma, and multicentric Castlemans disease. Although HHV-8 infection appears to be necessary, it may not be sufficient for development of KS without the involvement of other cofactors. One potentially important cofactor is HIV-1. HIV-1-infected cells produce HIV-1-related proteins and cytokines, both of which have been shown to promote growth of KS cells in vitro. Though HIV-1 is not absolutely necessary for KS development, KS is the most frequent neoplasm in AIDS patients, and AIDS-KS is recognized as a particularly aggressive form of the disease. To determine whether HIV-1 could participate in the pathogenesis of KS by modulating HHV-8 replication (rather than by inducing immunodeficiency), HIV-1-infected T cells were cocultured with the HHV-8-infected cell line, BCBL-1. The results demonstrate soluble factors produced by or in response to HIV-1-infected T cells induced HHV-8 replication, as determined by production of lytic phase mRNA transcripts, viral proteins, and detection of progeny virions. By focusing on cytokines produced in the coculture system, several cytokines known to be important in growth and proliferation of KS cells in vitro, particularly Oncostatin M, hepatocyte growth factor/scatter factor, and interferon-gamma, were found to induce HHV-8 lytic replication when added individually to BCBL-1 cells. These results suggest specific cytokines can play an important role in the initiation and progression of KS through reactivation of HHV-8. Thus, HIV-1 may participate more directly than previously recognized in KS by promoting HHV-8 replication and, hence, increasing local HHV-8 viral load.

Notch-independent regulation of Hes-1 expression by c-Jun N-terminal kinase signaling in human endothelial cells

Our laboratory has recently demonstrated constitutive activation of the Notch signaling pathway in Kaposis sarcoma tumor cells. As endothelial cells (EC) are believed to be the progenitor of these tumor cells, this study was designed to examine the effect of Notch activation on normal human EC. Recent reports suggest Notch activation induces EC growth arrest, and that this growth arrest may be linked to the establishment or maintenance of EC quiescence, the phenotype seen in contact-inhibited EC lining the vasculature. To gain further insight into Notch activation and quiescence, we first confirmed that Notch activation induced EC growth arrest. Next, we examined Notch activation in confluent, growth arrested EC (mimicking the cells lining the vasculature). In contrast to previous reports, we found confluent EC possess lower levels of activated Notch compared to proliferating control cells. Interestingly, these cells express elevated levels of Hes-1 protein (an immediate downstream target of Notch signaling) despite decreased Notch activation. Under these conditions, Hes-1 expression was mediated, at least in part, by a Notch-independent mechanism involving c-jun N-terminal protein kinase (JNK) signaling. This is the first report, to our knowledge, that JNK signaling can modulate Hes-1 expression in a Notch-independent manner.

The Helix-Loop-Helix Protein Id-1 Delays Onset of Replicative Senescence in Human Endothelial Cells

Id proteins are negative regulators of basic helix–loop–helix transcription factors, which are critical for expression of genes associated with cellular differentiation. Previous studies have shown that overexpression of Id-1 delays cellular senescence in several cell types, including fibroblasts, mammary epithelial cells, and keratinocytes. Although previous studies have demonstrated the expression of Id-1 in endothelium, the regulation of Id-1 has not been studied in these cells. In this report, a retroviral vector was used to overexpress Id-1 in human endothelial cells. Sustained expression of Id-1 resulted in a 2- to 3-fold increase in the total number of population doublings (replicative capacity) of the cells compared with vector-treated controls, which correlated with low levels of p16, p21, and p27 expression. The cells, however, were not immortalized and did eventually undergo senescence despite elevated Id-1 levels. Senescence was characterized by a dramatic increase in p16, but not p21 and p27. Under these experimental conditions, telomerase activity was not detected and the telomeres became progressively shorter with time. These results demonstrate the importance of Id-1 in endothelial cell proliferation and indicate that Id-1 represses p16 expression, resulting in delayed senescence. These findings may have implications in the development of endothelial cell–derived tumors.

Characterization of Uptake and Internalization of Exosomes by Bladder Cancer Cells

Bladder tumors represent a special therapeutic challenge as they have a high recurrence rate requiring repeated interventions and may progress to invasive or metastatic disease. Exosomes carry proteins implicated in bladder cancer progression and have been implicated in bladder cancer cell survival. Here, we characterized exosome uptake and internalization by human bladder cancer cells using Amnis ImageStreamX, an image cytometer. Exosomes were isolated by ultracentrifugation from bladder cancer culture conditioned supernatant, labeled with PKH-26, and analyzed on the ImageStreamX with an internal standard added to determine concentration. Exosomes were cocultured with bladder cancer cells and analyzed for internalization. Using the IDEAS software, we determined exosome uptake based on the number of PKH-26+ spots and overall PKH-26 fluorescence intensity. Using unlabeled beads of a known concentration and size, we were able to determine concentrations of exosomes isolated from bladder cancer cells. We measured exosome uptake by recipient bladder cancer cells, and we demonstrated that uptake is dose and time dependent. Finally, we found that uptake is active and specific, which can be partially blocked by heparin treatment. The characterization of cellular uptake and internalization by bladder cancer cells may shed light on the role of exosomes on bladder cancer recurrence and progression.

Molecular mechanisms of replicative senescence in endothelial cells

As human somatic cells age, they stop replicating and enter an irreversible state of growth arrest known as replicative senescence. Senescent cells are viable, metabolically active, and display altered gene and protein expression compared to proliferating cells. Endothelial cells, both in vitro and in vivo, are known to undergo senescence. As endothelial cells are a critical component of the vasculature, senescence of these cells can have a significant impact of vascular integrity, function, and overall homeostasis. This review will summarize recent work to understand the molecular mechanisms of endothelial cell senescence and the resulting alterations in gene/protein expression in these cells. Endothelial cell senescence will then be discussed in the context of disease development with a focus on atherosclerosis, an important age-associated disease of the vasculature.