Benign and low-grade superficial endothelial cell neoplasms in the molecular era.

Vascular tumors are the most common mesenchymal neoplasms of the skin and subcutis, and they encompass a heterogeneous group with diverse clinical, histological, and molecular features, as well as biological behavior. Over the past two decades, molecular studies have enabled the identification of pathogenic recurrent genetic alterations that can be used as additional data points to support the correct classification of these lesions. The purpose of this review is to summarize the available data related to superficially located benign and low-grade vascular neoplasms and to highlight recent molecular advances with the role of surrogate immunohistochemistry to target pathogenic proteins as diagnostic biomarkers.

Mitochondria as Target for Tumor Management of Hemangioendothelioma.

Aims: Hemangioendothelioma (HE) may be benign or malignant. Mouse hemangioendothelioma endothelial (EOMA) cells are validated to study mechanisms in HE. This work demonstrates that EOMA cells heavily rely on mitochondria to thrive. Thus, a combination therapy, including weak X-ray therapy (XRT, 0.5 Gy) and a standardized natural berry extract (NBE) was tested. This NBE is known to be effective in managing experimental HE and has been awarded with the Food and Drug Administration Investigational New Drug (FDA-IND) number 140318 for clinical studies on infantile hemangioma. Results: NBE treatment alone selectively attenuated basal oxygen consumption rate of EOMA cells. NBE specifically sensitized EOMA, but not murine aortic endothelial cells to XRT-dependent attenuation of mitochondrial respiration and adenosine triphosphate (ATP) production. Combination treatment, selectively and potently, influenced mitochondrial dynamics in EOMA cells such that fission was augmented. This was achieved by lowering of mitochondrial sirtuin 3 (SIRT3) causing increased phosphorylation of AMP-activated protein kinase (AMPK). A key role of SIRT3 in loss of EOMA cell viability caused by the combination therapy was evident when pyrroloquinoline quinone, an inducer of SIRT3, pretreatment rescued these cells. Innovation and Conclusion: Mitochondria-targeting NBE significantly extended survival of HE-affected mice. The beneficial effect of NBE in combination with weak X-ray therapy was, however, far more potent with threefold increase in murine survival. The observation that safe natural products may target tumor cell mitochondria and sharply lower radiation dosage required for tumor management warrants clinical testing.

Immunohistochemical Analysis of mTOR Pathway-Related Proteins in Kaposiform Hemangioendothelioma.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors have been shown to have excellent effects in the management of kaposiform hemangioendothelioma (KHE); however, the mechanism of action is unclear. This study identified the expressions of mTOR pathway-related proteins in different vascular tumors to provide insight into the pathogenesis of KHE. METHODS: We retrospectively reviewed the pathologic specimens of 30 patients (KHE, 15; tufted angioma [TA], 5; infantile hemangioma [IH], 5; and lymphatic malformation [LM], 5). The immunohistochemical expression of mTOR-related proteins tuberous sclerosis complex 2 (TSC2), phosphatase and tensin homologue (PTEN), phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1), phosphorylated mTOR (p-mTOR), and phosphorylated ribosomal protein S6 kinase B1 (p-P70S6K) were analyzed using Image-Pro Plus software. KHE had the following pattern of expression in the spindle vascular endothelial cells: TSC2 (-); PTEN (-); p-4EBP1 (+); p-mTOR (+); and p-P70S6K (+). RESULTS: All 3 patients treated with sirolimus had good responses. The TA results were similar to KHE with no significant differences (p-4EBP1: p = 0.0687; p-mTOR: p = 0.0832). The expressions of TSC2, PTEN, p-4EBP1, p-mTOR, and p-P70S6K were negative or weakly positive in IH with a statistically significant difference compared to KHE (p-4EBP1: p < 0.001; p-mTOR: p < 0.001; p-P70S6K: p < 0.001). LM had no significant differences when compared to KHE. CONCLUSIONS: The absence of TSC2 and PTEN caused abnormal activation of the mTOR signaling pathway and may be involved in the pathogenesis of KHE. The expression of mTOR-related proteins in TA and LM was similar to KHE, unlike IH. The KHE pattern of expression [PTEN (-), TSC2 (-), p-mTOR (+), p-P70S6K (+), and p-4EBP1 (+)] suggested that sirolimus may be a good therapeutic choice.

Molecular characterization of hepatic epithelioid hemangioendothelioma reveals alterations in various genes involved in DNA repair, epigenetic regulation, signaling pathways, and cell cycle control.

Epithelioid hemangioendotheliomas (EHE) of the liver are rare, low-malignant vascular tumors whose molecular pathogenesis is incompletely understood. The diagnosis of EHE is challenging, and the course of the disease can be highly variable. Therapeutic options for EHE are limited, including resection of primary and metastatic tumors, organ transplantation and rather ineffective systemic approaches. Driver mutations have been reported (fusion transcripts of either YAP-TFE3 or WWTR1-CAMTA1) but comprehensive molecular profiling has not been performed. Our aim was to molecularly characterize hepatic EHE to identify new molecular targets. Eight primary hepatic EHE were analyzed by next-generation sequencing using a 409-gene panel. The majority of primary hepatic EHE revealed a low number of mutations. Genes that were mutated primarily are involved in DNA repair, epigenetic regulation, signaling pathways and cell cycle control, indicating that EHE present with mutations in various functions. Although only detecting a low mutation rate, a comparison with comprehensive databases (target db V3) revealed mutations in five genes with putative therapeutical options. Therefore, our findings help to shed light on the molecular background of EHE and might pave the way to new therapeutic approaches.

Signaling pathways and inhibitors of cells from patients with kaposiform lymphangiomatosis.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) is a rare lymphatic anomaly with significant morbidity and mortality. KLA is characterized by diffuse multifocal lesions comprised of focal areas of "kaposiform" spindled cells accompanying malformed lymphatic channels. The goal of this study was to identify activated signaling pathways in cells isolated from three KLA patients for the purpose of testing new therapies. PROCEDURE: Cells were obtained from the lungs of one patient isolated at autopsy and the spleen of two patients removed in surgery due to disease complications. A protein kinase array was performed on the KLA cell lysates and normal lymphatic endothelial cells. RESULTS: Higher activation of key signaling pathways in the KLA cells, including PRAS40, AKT1/2/3, and ERK-1/2, was identified by protein kinase array and confirmed by Western blot analysis. This indicated a role for highly activated PI3K-AKT and MAPK-ERK-1/2 signaling pathways in KLA cells. Cell proliferation studies assessed PI3K inhibitors (LY294002; BYL719), AKT inhibitor ARQ092, mTOR inhibitor rapamycin, and MAPK inhibitor U0126. These studies demonstrated that PI3K-AKT-mTOR and MAPK signaling are important mediators of KLA cell proliferation. BYL719 and rapamycin were more effective at inhibiting KLA cell proliferation than U0126. CONCLUSIONS: Our studies using cells from KLA patient lesions demonstrate that these cells are highly proliferative and the PI3K-AKT-mTOR and MAPK pathways are promising therapeutic targets. Development and clinical trials of PI3K, AKT, and MAPK inhibitors for cancer treatment and the data in this study lend support for early clinical trials assessing the efficacy of these inhibitors in KLA patients.

Proliferative Cells From Kaposiform Lymphangiomatosis Lesions Resemble Mesenchyme Stem Cell-like Pericytes Defective in Vessel Formation.

Kaposiform lymphangiomatosis (KLA) is a vascular anomaly featuring lymphatic expansion. It has no known cause, no effective treatment, and is associated with high morbidity. Proliferative cells from 3 KLA patient lesions were characterized relative to adiopose-derived mesenchyme stem cells (ADSCs) and cells derived from a patient with the related disease kaposiform hemangioendothelioma (KHE). KLA cells variably expressed markers of mesenchyme stem cells (CD73, CD90, CD105, CD146) and lacked endothelial cell markers (CD31, CD34) as determined by flow cytometry. They expressed markers of vascular pericytes (neural/glial antigen 2, alpha-smooth muscle actin, platelet-derived growth factor-beta receptor, and CXCL12) as determined by quantitative reverse transcription polymerase chain reaction. Lesion cells transcribed vascular markers VEGFC and VEGFD, as well as VCAM-1, the latter of which was confirmed by flow cytometry, consistent with angiogenic MSC-like pericytes. Furthermore, conditioned medium from each was shown to promote the proliferation of growth factor-starved lymphatic endothelial cells. Unlike kaposiform hemangioendothelioma-derived MSC-like pericytes and ADSCs, KLA isolates were defective in support of vascular network formation in co-cultures with either vascular or lymphatic endothelial cells. Genetic analysis by whole exome sequencing revealed novel variant alleles in 2 populations of KLA cells (BAD, TSC1) that may bear on aberrant pericyte growth and function.

Integrin ß3/Akt signaling contributes to platelet-induced hemangioendothelioma growth.

Hemangioendothelioma (HE) is a type of angiomatous lesions that features endothelial cell proliferation. Understanding the mechanisms orchestrating HE angiogenesis can provide therapeutic insights. It has been shown that platelets can support normal and malignant endothelial cells during angiogenesis. Using the mouse endothelial-derived EOMA cell line as a model of HE, we explored the regulatory effect of platelets. We found that platelets stimulated EOMA proliferation but did not mitigate apoptosis. Furthermore, direct platelet-EOMA cell contact was required and the proliferation was mediated via integrin ß3/Akt signaling in EOMA cells. SiRNA knockdown of integrin ß3 and inhibition of Akt activity significantly abolished platelet-induced EOMA cell proliferation in vitro and tumor development in vivo. These results provide a new mechanism by which platelets support HE progression and suggest integrin ß3 as a potential target to treat HE.

Composite hemangioendothelioma with neuroendocrine marker expression: an aggressive variant.

Aberrant expression of neuroendocrine markers is extremely rare in endothelial neoplasms, with only a single report describing three cases. Although originally classified as conventional angiosarcoma, further assessment of these tumors revealed a strikingly composite morphology composed of retiform and epithelioid elements reminiscent of composite hemangioendothelioma, a rare subtype of hemangioendothelioma. To further investigate these findings, available materials from 11 morphologically distinctive endothelial tumors showing neuroendocrine marker expression were retrieved from our archives. Immunohistochemistry for CD31, CD34, FLI-1, synaptophysin, chromogranin, D2-40, ERG, keratin (OSCAR), and CAMTA1 was performed. Total RNA from five cases were extracted and subjected to whole transcriptome sequencing. Clinical follow-up was obtained. These tumors were found to arise in five males and six females in patients from 9 to 55 years in age (median 47 years). They arose both in superficial (wrist, ankle, scalp, hip, and foot) and deep (periaortic tissues, C5 vertebra, pulmonary vein, and liver) locations. All contained elongated, retiform vascular channels lined by hyperchromatic 'hobnail' endothelial cells and a solid growth of uniform epithelioid cells reminiscent of epithelioid hemangioendothelioma. Hemangioma-like foci also lined by hobnail endothelial cells were frequently present. Mitotic activity was typically <1/10 HPF, and necrosis or areas of conventional angiosarcoma was absent. The results of immunohistochemistry were: CD31 (10/10), FLI-1 (10/10), ERG (9/9), CD34 (5/10), D2-40 (7/10), synaptophysin (11/11), chromogranin A (1/11), CD56 (5/11), keratin (0/11), and CAMTA1 (0/6). Sequencing analysis showed one case with PTBP1-MAML2 and one case with EPC1-PHC2 fusion transcripts; fusion transcripts were not identified in the remaining cases. Follow-up (8 cases) revealed local recurrence in one patient and metastatic spread in four individuals (bone, lung, liver, and brain). One person died of disease. Although the morphological features of these tumors are characteristic of composite hemangioendothelioma, this distinctive subset with neuroendocrine differentiation more often involves deep locations and displays more aggressive behavior than typically described in other cases of composite hemangioendothelioma.

Nanoformulations for dimethyl fumarate: Physicochemical characterization and in vitro/in vivo behavior.

Dimethyl fumarate has been demonstrated useful in relapsing remitting multiple sclerosis treatment (Tecfidera®). Nevertheless, since Tecfidera® capsules induce flushing, gastro-intestinal events and other more serious drawbacks, in this investigation a nanoparticle based system to be administered by an alternative way is proposed. In particular this study describes the preparation and characterization of dimethyl fumarate-containing solid lipid nanoparticles (SLN). Namely SLN based on tristearin, tristearin SLN treated with polysorbate 80 and cationic SLN constituted of tristearin in mixture with dimethyldioctadecylammonium chloride were investigated. The effect of the presence of dimethyl fumarate, functionalization by polysorbate 80 and dimethyldioctadecylammonium chloride was studied on morphology and dimensional distribution of SLN, by photon correlation spectroscopy and cryogenic transmission electron microscopy. Dimethyl fumarate release from SLN, studied by Franz cell, evidenced a Fickian dissolutive type kinetic in the case of SLN treated by polysorbate 80. Moreover fluorescent SLN were produced and characterized in order to investigate their in vitro permeability and in vivo biodistribution in mice. An in vitro study of fluorescent SLN permeability performed through a model of mouse brain microvascular endothelial cells, indicated that cationic SLN displayed higher permeability values with respect to neutral SLN and SLN treated by polysorbate 80. Biodistribution of polysorbate 80 treated SLN was studied by fluorescent imaging after intraperitoneal or intranasal administration in mice. The in vivo images indicate that polysorbate 80 treated SLN were able to reach the brain, even if they prevalently accumulated in liver and spleen, especially by intraperitoneal route.

Phytochemical Inhibition of Multidrug Resistance Protein-1 as a Therapeutic Strategy for Hemangioendothelioma.

AIMS: Hemangiomas are endothelial cell tumors and the most common soft tissue tumors in infants. They frequently cause deformity and can cause death. Current pharmacologic therapies have high-risk side-effect profiles, which limit the number of children who receive treatment. The objectives of this work were to identify the mechanisms through which standardized berry extracts can inhibit endothelial cell tumor growth and test these findings in vivo. RESULTS: EOMA cells are a validated model that generates endothelial cell tumors when injected subcutaneously into syngeneic (129P/3) mice. EOMA cells treated with a blend of powdered natural berry extracts (NBE) significantly inhibited activity of multidrug resistance protein-1 (MRP-1) compared to vehicle controls. This resulted in nuclear accumulation of oxidized glutathione (GSSG) and apoptotic EOMA cell death. When NBE-treated EOMA cells were injected into mice, they generated smaller tumors and had a higher incidence of apoptotic cell death compared to vehicle-treated EOMA cells as demonstrated by immunocytochemistry. Kaplan-Meier survival curves for tumor-bearing mice showed that NBE treatment significantly prolonged survival compared to vehicle-treated controls. INNOVATION: These are the first reported results to show that berry extracts can inhibit MRP-1 function that causes apoptotic tumor cell death by accumulation of GSSG in the nucleus of EOMA cells where NADPH oxidase is hyperactive and causes pathological angiogenesis. CONCLUSIONS: These findings indicate that berry extract inhibition of MRP-1 merits consideration and further investigation as a therapeutic intervention and may have application for other cancers with elevated MRP-1 activity. Antioxid. Redox Signal. 26, 1009-1019.

The Epithelial Sodium Channel and the Processes of Wound Healing.

The epithelial sodium channel (ENaC) mediates passive sodium transport across the apical membranes of sodium absorbing epithelia, like the distal nephron, the intestine, and the lung airways. Additionally, the channel has been involved in the transduction of mechanical stimuli, such as hydrostatic pressure, membrane stretch, and shear stress from fluid flow. Thus, in vascular endothelium, it participates in the control of the vascular tone via its activity both as a sodium channel and as a shear stress transducer. Rather recently, ENaC has been shown to participate in the processes of wound healing, a role that may also involve its activities as sodium transporter and as mechanotransducer. Its presence as the sole channel mediating sodium transport in many tissues and the diversity of its functions probably underlie the complexity of its regulation. This brief review describes some aspects of ENaC regulation, comments on evidence about ENaC participation in wound healing, and suggests possible regulatory mechanisms involved in this participation.

PF573,228 inhibits vascular tumor cell growth, migration as well as angiogenesis, induces apoptosis and abrogates PRAS40 and S6RP phosphorylation.

PF573,228 is a compound that targets focal adhesion kinase (FAK), a non-receptor protein kinase, which is over-expressed in various tumors. The aim of this study was to evaluate the effects of PF573,228 on the cells derived from mouse vascular tumors, namely, endothelioma cells. The treatment of endothelioma cells with PF573,228 reduced their growth with an IC50 of approximately 4.6 µmol L-1 and inhibited cell migration with an IC50 of about 0.01 µmol L-1. Microscopic studies revealed morphological attributes of apoptosis. These observations were confirmed by ELISA, which showed increased caspase-3 activity. PF573,228 also inhibited angiogenesis in a dose-dependent manner, with an IC50 of approximately 3.7 µmol L-1, and abrogated the phosphorylation of cell survival proteins, proline-rich Akt substrate (PRAS40) and S6 ribosomal protein (S6RP). Array data further revealed that PF573,228 induced caspase-3 activation, thus promoting apoptosis. Since all the processes inhibited by PF573,228 provide important support to tumor survival and progression, the drug may have a potential role in the treatment of vascular tumors.

Developing a Nanoparticle-Delivered High-Efficacy Treatment for Infantile Hemangiomas Using a Mouse Hemangioendothelioma Model.

BACKGROUND: Current treatments for infantile hemangiomas have unpredictable outcomes. The authors' aim was to develop a nanoporphyrin-delivered, high-efficacy treatment for infantile hemangiomas using a mouse hemangioendothelioma model. METHODS: The authors injected mouse hemangioendothelioma cells intradermally to axillary regions of 5-week-old, female, nude mice (n = 19) to induce hemangioendothelioma growth. They documented nanoporphyrin accumulation in hemangioendotheliomas using positron emission tomography. For the treatment study, the authors randomized hemangioendothelioma-bearing nude mice (n = 9) into three groups (n = 3 each). Group I received only saline injections. Group II received only laser treatment after saline injection, and group III received laser treatment after nanoporphyrin injection through the tail vein. The authors followed up the treatment response with digital caliper measurements. RESULTS: Hemangioendotheliomas started to grow approximately 1 week after inoculation and resembled infantile hemangiomas histologically. Nanoporphyrin uptake in hemangioendotheliomas was 19.7 ± 2.2, 16.7 ± 2.02, 8.4 ± 0.3, and 4.9 ± 0.6 percent injected dose per gram of tissue at 3, 6, 24, and 48 hours after injection, respectively. Nanoporphyrin uptake was significantly higher than in blood at 24 and 48 hours after injection (p < 0.05). Results of ex vivo biodistribution study were consistent with positron emission tomographic imaging. Hemangioendotheliomas in group III started to regress 1 day after the treatment and disappeared totally by day 21. The difference between tumor volumes in group III and other groups was significant on days 17 and 21 (p < 0.05). CONCLUSIONS: Nanoporphyrin accumulated in hemangioendotheliomas at high concentrations, enabling a high-efficacy photodynamic therapy. Given the similarities between hemangioendotheliomas and infantile hemangiomas, this treatment potentially can be a high-efficacy treatment for infantile hemangiomas.

Heat stress prevents lipopolysaccharide-induced apoptosis in pulmonary microvascular endothelial cells by blocking calpain/p38 MAPK signalling.

Pulmonary microvascular endothelial cells (PMECs) injury including apoptosis plays an important role in the pathogenesis of acute lung injury during sepsis. Our recent study has demonstrated that calpain activation contributes to apoptosis in PMECs under septic conditions. This study investigated how calpain activation mediated apoptosis and whether heat stress regulated calpain activation in lipopolysaccharides (LPS)-stimulated PMECs. In cultured mouse primary PMECs, incubation with LPS (1 µg/ml, 24 h) increased active caspase-3 fragments and DNA fragmentation, indicative of apoptosis. These effects of LPS were abrogated by pre-treatment with heat stress (43 °C for 2 h). LPS also induced calpain activation and increased phosphorylation of p38 MAPK. Inhibition of calpain and p38 MAPK prevented apoptosis induced by LPS. Furthermore, inhibition of calpain blocked p38 MAPK phosphorylation in LPS-stimulated PMECs. Notably, heat stress decreased the protein levels of calpain-1/2 and calpain activities, and blocked p38 MAPK phosphorylation in response to LPS. Additionally, forced up-regulation of calpain-1 or calpain-2 sufficiently induced p38 MAPK phosphorylation and apoptosis in PMECs, both of which were inhibited by heat stress. In conclusion, heat stress prevents LPS-induced apoptosis in PMECs. This effect of heat stress is associated with down-regulation of calpain expression and activation, and subsequent blockage of p38 MAPK activation in response to LPS. Thus, blocking calpain/p38 MAPK pathway may be a novel mechanism underlying heat stress-mediated inhibition of apoptosis in LPS-stimulated endothelial cells.

Phosphoinositide 3-kinase/Akt pathway is involved in pingyangmycin­induced growth inhibition, apoptosis and reduction of invasive potential in EOMA mouse hemangioendothelioma cells.

Pingyangmycin (PYM), a glycopeptide antibiotic, has been recommended as a stand treatment for hemangioma. However, the underlying mechanisms of its anti­tumor effects have remained elusive. The purpose of the present study was to explore the effects of PYM on the biological behavior of the EOMA mouse hemangioendothelioma cell line and investigate the possible mechanisms. The effects of PYM on EOMA cell viability were determined by an MTT assay, apoptosis was evaluated by Annexin V/propidium iodide staining and flow cytometric analysis, and cell invasion ability was determined using a Transwell invasion assay. In order to investigate the underlying mechanism of action of PYM, the expression of angiogenic signaling proteins was determined by western blot analysis. PYM treatment (0.5­500 µg/ml) inhibited cell growth in a time- and dose­dependent manner. PYM at 100 µg/ml significantly induced apoptosis and reduced the invasive ability of EOMA cells. Effects of PYM on cell viability, apoptosis and invasion ability were completely blocked by co­treatment with phosphoinositide 3­kinase (PI3K) activator insulin­like growth factor­1 (IGF­1). Furthermore, treatment with PYM reduced the expression of PI3K and phosphorylated Akt. In conclusion, the present study indicated that the PI3K/Akt pathway is likely to be involved in the anti-cancer effects of PYM on EOMA cells.

Clinicopathological features of Kaposiform hemangioendothelioma.

Kaposiform hemangioendothelioma (KHE), an intermediate tumor of endothelial origin in childhood, is often associated with Kasabach-Merritt phenomenon (KMP). In this study, 22 cases of KHE were immunochemically studied for CD31, CD34, ERG, smooth muscle actin (SMA), D240, GLUT1 and Ki67. The patients (15 males and 7 females) ranged in age from 13 days to 7 years (median, 2 mo). Lesion developed on the extremities/joint (12 cases), chest/abdominal wall (6 cases), head/neck (4 cases), and presented both superficial and deep soft tissue. The superficial change was commonly enlarging cutaneous lesion with ill-defined red to purple indurated plaque. 15 of the 22 cases (68%) developed KMP, with consumptive thrombocytopenia or bleeding complications. Tumors consisted of infiltrating nodules of fascicles of spindleshaped endothelial cells and slitlike vascular channels with irregular tumor margins. On immunohistochemistry (IHC), endothelial cells were diffusely positive for CD34, CD31 and ERG but negative for GLUT1, and the peripheral area of proliferative capillaries were markedly positive for D240. Adjuvant medical therapy and sclerotherapy were prepared for the tumor and the associated KMP, and then all patients were treated by complete surgical excision. Follow-up information was available in 22 patients (8 to 26 months, mean 15 mo), and indicated that 1 died of multiple organ failure and 21 were alive without residual disease. In conclusion, our results suggest that KHE can occur in the embryonic period, and patients with KMP often have earlier onset time and larger lesional size. KHE patients given with adjuvant corticosteroids and urea injection and complete resection rarely relapse.

Adhesion molecule-mediated hippo pathway modulates hemangioendothelioma cell behavior.

Hemangioendotheliomas are categorized as intermediate-grade vascular tumors that are commonly localized in the lungs and livers. The regulation of this tumor cell's proliferative and apoptotic mechanisms is ill defined. We recently documented an important role for Hippo pathway signaling via endothelial cell adhesion molecules in brain microvascular endothelial cell proliferation and apoptosis. We found that endothelial cells lacking cell adhesion molecules escaped from contact inhibition and exhibited abnormal proliferation and apoptosis. Here we report on the roles of adherens junction molecule modulation of survivin and the Hippo pathway in the proliferation and apoptosis of a murine hemangioendothelioma (EOMA) cell. We demonstrated reduced adherens junction molecule (CD31 and VE-cadherin) expression, increased survivin and Ajuba expression, and a reduction in Hippo pathway signaling resulting in increased proliferation and decreased activation of effector caspase 3 in postconfluent EOMA cell cultures. Furthermore, we confirmed that YM155, an antisurvivin drug that interferes with Sp1-survivin promoter interactions, and survivin small interference RNA (siRNA) transfection elicited induction of VE-cadherin, decreased Ajuba expression, increased Hippo pathway and caspase activation and apoptosis, and decreased cell proliferation. These findings support the importance of the Hippo pathway in hemangioendothelioma cell proliferation and survival and YM155 as a potential therapeutic agent in this category of vascular tumors.