Peter J. Polverini

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.

Bcl-2 Acts in a Proangiogenic Signaling Pathway through Nuclear Factor-κB and CXC Chemokines

Vascular endothelial growth factor (VEGF) induces expression of Bcl-2 in tumor-associated microvascular endothelial cells. We have previously reported that up-regulated Bcl-2 expression in microvascular endothelial cells is sufficient to enhance intratumoral angiogenesis and to accelerate tumor growth. We initially attributed these results to Bcl-2-mediated endothelial cell survival. However, in recent experiments, we observed that conditioned medium from Bcl-2-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-2) is sufficient to induce potent neovascularization in the rat corneal assay, whereas conditioned medium from empty vector controls (HDMEC-LXSN) does not induce angiogenesis. These results cannot be attributed to the role of Bcl-2 in cell survival. To understand this unexpected observation, we did gene expression arrays that revealed that the expression of the proangiogenic chemokines interleukin-8 (CXCL8) and growth-related oncogene-alpha (CXCL1) is significantly higher in HDMEC exposed to VEGF and in HDMEC-Bcl-2 than in controls. Inhibition of Bcl-2 expression with small interfering RNA-Bcl-2, or the inhibition of Bcl-2 function with small molecule inhibitor BL-193, down-regulated CXCL8 and CXCL1 expression and caused marked decrease in the angiogenic potential of endothelial cells without affecting cell viability. Nuclear factor-kappaB (NF-kappaB) is highly activated in HDMEC exposed to VEGF and HDMEC-Bcl-2 cells, and genetic and chemical approaches to block the activity of NF-kappaB down-regulated CXCL8 and CXCL1 expression levels. These results reveal a novel function for Bcl-2 as a proangiogenic signaling molecule and suggest a role for this pathway in tumor angiogenesis.

Bcl-2 protects endothelial cells against γ-radiation via a raf-MEK-ERK-survivin signaling pathway that is independent of cytochrome c release

The Bcl-2 oncoprotein is a potent inhibitor of apoptosis and is overexpressed in a wide variety of malignancies. Until recently, it was generally accepted that Bcl-2 primarily mediates its antiapoptotic function by regulating cytochrome c release from mitochondria. However, more recent studies have shown that Bcl-2 is present on several intracellular membranes and mitochondria may not be the only site where Bcl-2 exercises its survival function. In this study, we investigated if Bcl-2 can protect endothelial cells against gamma-radiation by a cytochrome c-independent signaling pathway. Human dermal microvascular endothelial cells (HDMEC), when exposed to gamma-radiation, exhibited a time-dependent activation of caspase-3 that was associated with increased cytochrome c release from mitochondria. Bcl-2 expression in endothelial cells (HDMEC-Bcl-2) significantly inhibited irradiation-induced caspase-3 activation. However, Bcl-2-mediated inhibition of caspase-3 was significantly reversed by inhibition of the Raf-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway. Interestingly, caspase-3 activation in HDMEC-Bcl-2 cells was not associated with cytochrome c release. We also observed that endothelial cell Bcl-2 expression significantly increased the expression of survivin and murine double minute-2 (Mdm2) via the Raf-MEK-ERK pathway. Endothelial cells expressing Bcl-2 also inhibited gamma-radiation-induced activation of p38 MAPK and p53 accumulation. Inhibition of p53 accumulation in HDMEC-Bcl-2 could be due to the enhanced expression of Mdm2 in these cells. Taken together, these results show three mechanisms by which Bcl-2 may mediate endothelial cell cytoprotection independently of cytochrome c release: (a) increased survivin expression, (b) inhibition of p53 accumulation, and (c) inhibition of p38 MAPK.

Arsenic trioxide enhances the therapeutic efficacy of radiation treatment of oral squamous carcinoma while protecting bone

Therapeutic radiation is commonly used in the treatment of squamous cell carcinoma of the oral cavity and pharynx. Despite the proven efficacy of this form of anticancer therapy, high-dose radiation treatment is invariably associated with numerous unwanted side effects. This is particularly true for bone, in which radiation treatment often leads to osteoradionecrosis. The aim of this study was to investigate if treatment with arsenic trioxide (As2O3) could enhance the antitumor effect of radiotherapy whereas minimizing the destructive effects of radiation on bone. As2O3 treatment induced a dose-dependent (1–20 μmol/L) inhibition of endothelial and tumor cell (OSCC-3 and UM-SCC-74A) survival and significantly enhanced radiation-induced endothelial cell and tumor cell death. In contrast, As2O3 treatment (0.5–7.5 μmol/L) induced the proliferation of osteoblasts and also protected osteoblasts against radiation-induced cell death. Furthermore, As2O3 treatment was able to significantly enhance radiation-induced inhibition of endothelial cell tube formation and tumor cell colony formation. To test the effectiveness of As2O3 and radiation treatment in vivo, we used a severe combined immunodeficiency mouse model that has a bone ossicle and tumor growing side by side subcutaneously. Animals treated with As2O3 and radiation showed a significant inhibition of tumor growth, tumor angiogenesis, and tumor metastasis to the lungs as compared with As2O3 treatment or radiation treatment alone. In contrast, As2O3 treatment protected bone ossicles from radiation-induced bone loss. These results suggest a novel strategy to enhance the therapeutic efficacy of radiation treatment while protecting bone from the adverse effects of therapeutic radiation. [Mol Cancer Ther 2008;7(7):2060–9]

Endothelial cells expressing Bcl-2 promotes tumor metastasis by enhancing tumor angiogenesis, blood vessel leakiness and tumor invasion

Metastatic spread of tumor cells to vital organs is the major cause of mortality in cancer patients. Bcl-2, a key antiapoptotic protein, is expressed at high levels in a number of human tumors. We have recently shown that Bcl-2 is also overexpressed in tumor-associated blood vessels in head-and-neck cancer patients. Interestingly, enhanced Bcl-2 expression in tumor blood vessels is directly correlated with metastatic status of these cancer patients. In addition, endothelial cells (ECs) expressing Bcl-2 showed increased production of interleukin-8 (IL-8) resulting in significantly enhanced tumor cell proliferation and tumor cell invasion. Therefore, we hypothesized that Bcl-2 expression in tumor-associated ECs may promote tumor metastasis by enhancing tumor cell invasiveness and release in the circulation. To test our hypothesis, we coimplanted tumor cells along with ECs expressing Bcl-2 (EC-Bcl-2) in the flanks of SCID mice. Our results demonstrate that incorporation of EC-Bcl-2 in primary tumors significantly enhanced tumor cell metastasis to lungs and this EC-Bcl-2-mediated tumor metastasis was independent of primary tumor size. In addition, Bcl-2-mediated tumor metastasis directly correlated with increased tumor angiogenesis. Bcl-2 expression in ECs also promoted transendothelial cell permeability, blood vessel leakiness and tumor cell invasion. EC-Bcl-2-mediated tumor cell proliferation and tumor cell invasion were significantly mediated by IL-8. These results suggest that Bcl-2, when expressed at higher levels in tumor-associated ECs, may promote tumor metastasis by enhancing tumor angiogenesis, blood vessel leakiness and tumor cell invasiveness.

Differential angiogenic and proliferative activity of surgical and burn wound fluids.

BACKGROUND Invasive surgical wounds exhibit the rapid production of a robustly proangiogenic environment. To compare the immediate angiogenic environment of wounds of different types, the angiogenic activity of fluid derived from burn injuries and wounds confined to the dermis was examined and compared with that of deeper surgical wounds. METHODS The angiogenic activity of surgical wound fluid (SWF) (n = 7), skin graft wound fluid (SGF) (n = 3), and burn wound fluid (BWF) (n = 4) was assessed by measuring endothelial cell (EC) proliferative activity, EC chemotactic activity, and angiogenic activity in the rat corneal assay. The fibroblast growth factor-2 (FGF-2) level of each wound fluid was determined by enzyme-linked immunosorbent assay. RESULTS SWF exhibited significant EC proliferative activity, SGF exhibited intermediate activity, and BWF displayed no EC proliferative activity. Seventy-one percent of SWF samples, 33% of SGF, and 0% of BWF contained significant EC chemotactic activity. Each wound fluid sample that demonstrated significant chemotactic activity also evoked a positive corneal angiogenic response. SWF contained 914 +/- 170 pg/mL of FGF-2, whereas SGF and BWF contained just 164 +/- 54 pg/mL and 37 +/- 7 pg/mL of FGF-2, respectively. CONCLUSION The results suggest that injuries confined to the dermis, whether thermal or excisional, elicit a less robust initial angiogenic stimulus than deep surgical wounds.

Quantification of human angiogenesis in immunodeficient mice using a photon counting-based method

Testing new antiangiogenic drugs for cancer treatment requires the use of animal models, since stromal cells and extracellular matrices mediate signals to endothelial cells that cannot be fully reproduced in vitro. Most methods used for analysis of antiangiogenic drugs in vivo utilized histologic examination of tissue specimens, which often requires large sample sizes to obtain reliable quantitative data. Furthermore, these assays rely on the analysis of murine vasculature that may not be correlated with the responses of human endothelial cells. Here, we engineered human blood vessels in immunodeficient mice with human endothelial cells expressing luciferase, demonstrated that these cells line functional blood vessels, and quantified angiogenesis over time using a photon counting-based method. In a proof-of-principle experiment with PTK/ZK, a small molecule inhibitor of vascular endothelial growth factor (VEGF) tyrosine kinase receptors, a strong correlation was observed between the decrease in bioluminescence (9.12-fold) in treated mice and the actual decrease in microvessel density (9.16-fold) measured after retrieval of the scaffolds and immunohistochemical staining of endothelial cells. The method described here allows for quantitative and noninvasive investigation into the effects of anti-cancer drugs on human angiogenesis in a murine host.

C-X-C Chemokines and Lung Cancer Angiogenesis

Angiogenesis is an essential biologic event encountered in vertebrate animals (6,35,36,38,39,60,92). Embryonic development, the formation of inflammatory granulation tissue during wound healing, chronic inflammation, and the growth of malignant solid tumors represent physiologic and pathologic processes that are strictly dependent on neovascularization. The rate of normal capillary endothelial cell turnover in adults is typically measured in months or years (30,132). However, during wound repair and development of granulation tissue, resting endothelial cells become activated, leading to proteolytic degradation of their basement membrane and surrounding extracellular matrix, migration, proliferation, and establishment of newly functioning capillaries within a matter of days (60). An important feature of wound-associated angiogenesis is that it is locally controlled and transient. As rapidly as neovascularization occurs, these new vessels virtually disappear, returning the tissue vasculature to a homeostatic environment. This abrupt termination of angiogenesis in the context of the resolution of wound repair supports the notion of two possible mechanisms of control. First, there is probably a marked reduction in the synthesis and/or elaboration of angiogenic mediators. Second, a simultaneous increase occurs in the levels of factors that inhibit neovascularization (15). In contrast to the precise regulation of angiogenesis that accompanies wound repair, dysregulation of angiogenesis can lead to an imbalance in overexpression of angiogenic and underexpression of angiostatic factors that contributes to the pathogenesis of solid tumor growth. Thus, the complement of positive and negative regulators of angiogenesis may vary among different physiologic and pathologic settings. The recognition of this dual mechanism of control is critical in order to gain insight into this complex process and to understand its significance in regulating net angiogenesis.

UM-HACC-2A: MYB-NFIB fusion-positive human adenoid cystic carcinoma cell line

OBJECTIVES Limited availability of validated human adenoid cystic carcinoma (ACC) cell lines has hindered the mechanistic understanding of the pathobiology of this malignancy and the development of effective therapies. The purpose of this work was to generate and characterize a human ACC cell line. MATERIAL AND METHODS Immediately after surgery, a tumor fragment from a minor salivary gland from the tongue of a female Caucasian was minced, dissociated, and a single cell suspension was plated in fibronectin-coated flasks. A culture medium containing bovine brain extract and rhEGF was optimized for these cells. Whole exome sequencing was used to evaluate the presence of MYB-NFIB translocation. RESULTS The University of Michigan-Human Adenoid Cystic Carcinoma (UM-HACC)-2A cells showed continuous growth in monolayers for at least 180 in vitro passages while maintaining epithelial morphology. Short-tandem repeat (STR) profiling confirmed a 100% match to patient DNA. Whole exome sequencing revealed the presence of the MYB-NFIB fusion in UM-HACC-2A cells, which was confirmed by PCR analysis. Western blots revealed high expression of epithelial markers (e.g. E-cadherin, EGFR, pan-cytokeratin) and proteins associated with ACC (e.g. c-Myb, p63). Developmental therapeutic studies showed that UM-HACC-2A cells were resistant to cisplatin (IC50 = 44.7 µM) while more responsive to paclitaxel (IC50 = 0.0006 µM). In a pilot study, we observed that UM-HACC-2A cells survived orthotopic transplantation into the submandibular gland. Notably, one of the mice injected with UM-HACC-2A cells exhibited lung metastasis after 6 months. CONCLUSION UM-HACC-2A is a MYB-NFIB fusion-positive ACC cell line that is suitable for mechanistic and developmental therapeutics studies.

Abstract 4993: IRE1-XBP1 branch of UPR is involved in tumor angiogenesis

The Unfolded Protein Response (UPR) is activated when the endoplasmic reticulum (ER) in tumor cells is subjected to environmental stress. We and others have previously shown that the eIF2α- PERK-ATF4 pathway, one of three of UPR pathways is involved in tumor angiogenesis by transcriptionally regulating the production of angiogenic mediators such as VEGF. However, role of other UPR pathways in tumor angiogenesis still remains to be elucidated. In this study we examined the role of the IRE1-XBP1 UPR pathway in promoting production of tumor-derived proangiogenic mediators. Upon ER stress IRE1 splices XBP1 into an active transcription factor that induces expression of several target genes. We used the gene-silencing approach in the oral squamous carcinoma cell line UM-SCC-81B to determine the effects of XBP1 knockdown on key angiogenic mediators. We were able to achieve over 80% knockdown of XBP1. mRNA levels of both EDEM1 and Grp78, downstream targets of XBP1 were also significantly inhibited confirming an efficient knockdown. UM-SCC-81B control cells as well as UM-SCC-81B-XBP1 knockdown cells were initially treated with low glucose (5mM) for 24 h to induce UPR. Next, mRNA and protein levels of VEGF were determined using Real-Time PCR and ELISA respectively. We observed a 45% decrease in VEGF mRNA levels and around 36% decrease in VEGF protein levels of UM-SCC-81B-XBP1 knockdown cells upon ER stress. Additionally, we detected a significant decrease in the transcription of several other key angiogenic mediators such as FGF2, IL6, IL8 and CTGF (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4993. doi:1538-7445.AM2012-4993