Human Angiosarcoma Research Articles

Abstract A31: Biallelic Dicer1 loss in endothelial cells drives angiosarcoma development

Abstract Angiosarcoma is an aggressive and highly metastatic vascular sarcoma with an extremely poor prognosis. Because of the rarity of the disease, the molecular drivers and optimal treatment strategies for patients remain unclear, highlighting the need for novel genetic and in vivo animal models. MicroRNAs are small RNAs that regulate gene expression and DICER1 is the RNase III endoribonuclease that processes pre-microRNAs into mature microRNAs. DICER1 and microRNAs are central to numerous cellular processes and are often dysregulated in many cancers, including sarcomas. Here we show that biallelic Dicer1 deletion with endothelial expression of aP2-Cre drives aggressive and metastatic angiosarcoma independent of other genetically engineered oncogenes or tumor suppressor loss. Angiosarcomas in aP2-Cre;Dicer1Flox/- mice histologically and genetically resemble human angiosarcoma, and we found microRNA-23 target genes enriched in mouse and human angiosarcoma. Because Ras mutations and Cdkn2a loss are common in angiosarcoma, we interrogated them as angiosarcoma modifiers. Interestingly, we found that both oncogenic LSL-KrasG12D and loss of conditional Cdkn2aFlox decreased tumor latency. In addition, aP2-Cre;LSL-KrasG12D;Cdkn2aFlox/Flox mice developed angiosarcomas, providing a model with Dicer1 expression intact to further study the function of microRNAs in angiosarcoma. Thus, these findings illustrate that Dicer1 can function as a traditional loss-of-function tumor-suppressor gene and provide simple and fully penetrant animal models for the study of angiosarcoma development and metastasis. Citation Format: Jason A. Hanna, Catherine J. Drummond, Matthew R. Garcia, Jonathan C. Go, David Finkelstein, Jerold E. Rehg, Mark E. Hatley. Biallelic Dicer1 loss in endothelial cells drives angiosarcoma development [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A31.

Mouse genetic background influences whether HrasG12V expression plus Cdkn2a knockdown causes angiosarcoma or undifferentiated pleomorphic sarcoma.

Soft tissue sarcomas are rare mesenchymal tumours accounting for 1% of adult malignancies and are fatal in approximately one third of patients. Two of the most aggressive and lethal forms of soft tissue sarcomas are angiosarcomas and undifferentiated pleomorphic sarcomas (UPS). To examine sarcoma-relevant molecular pathways, we employed a lentiviral gene regulatory system to attempt to generate in vivo models that reflect common molecular alterations of human angiosarcoma and UPS. Mice were intraveneously injected with MuLE lentiviruses expressing combinations of shRNA against Cdkn2a, Trp53, Tsc2 and Pten with or without expression of HrasG12V, PIK3CAH1047R or Myc. The systemic injection of an ecotropic lentivirus expressing oncogenic HrasG12V together with the knockdown of Cdkn2a or Trp53 was sufficient to initiate angiosarcoma and/or UPS development, providing a flexible system to generate autochthonous mouse models of these diseases. Unexpectedly, different mouse strains developed different types of sarcoma in response to identical genetic drivers, implicating genetic background as a contributor to the genesis and spectrum of sarcomas.

CD31 Expression Determines Redox Status and Chemoresistance in Human Angiosarcomas.

Purpose: Angiosarcomas are soft tissue sarcomas with endothelial differentiation and vasoformative capacity. Most angiosarcomas show strong constitutive expression of the endothelial adhesion receptor CD31/PECAM-1 pointing to an important role of this molecule. However, the biological function of CD31 in angiosarcomas is unknown.Experimental Design: The expression levels of CD31 in angiosarcoma cells and its effects on cell viability, colony formation, and chemoresistance were evaluated in human angiosarcoma clinical samples and in cell lines through isolation of CD31high and CD31low cell subsets. The redox-regulatory CD31 function linked to YAP signaling was determined using a CD31-blocking antibody and siRNA approach and was further validated in CD31-knockout endothelial cells.Results: We found that most angiosarcomas contain a small CD31low cell population. CD31low cells had lost part of their endothelial properties and were more tumorigenic and chemoresistant than CD31high cells due to more efficient reactive oxygen species (ROS) detoxification. Active downregulation of CD31 resulted in loss of endothelial tube formation, nuclear accumulation of YAP, and YAP-dependent induction of antioxidative enzymes. Addition of pazopanib, a known enhancer of proteasomal YAP degradation resensitized CD31low cells for doxorubicin resulting in growth suppression and induction of apoptosis.Conclusions: Human angiosarcomas contain a small aggressive CD31low population that have lost part of their endothelial differentiation programs and are more resistant against oxidative stress and DNA damage due to intensified YAP signaling. Our finding that the addition of YAP inhibitors can resensitize CD31low cells toward doxorubicin may aid in the rational development of novel combination therapies to treat angiosarcomas. Clin Cancer Res; 24(2); 460-73. ©2017 AACR.

Effects of DLC1 Deficiency on Endothelial Cell Contact Growth Inhibition and Angiosarcoma Progression.

Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined. We investigated the effects of shRNA-induced DLC1 depletion in endothelial cells. Cell growth was measured by 3H thymidine incorporation, IncuCyte imaging, and population doublings; cell death by cell cycle analysis; gene expression by Affimetrix arrays and quantitative polymerase chain reaction; NF-κB activity by reporter assays; and protein levels by immunoblotting and immunofluorescence staining. We tested Tanespimycin/17-AAG and Fasudil treatment in groups of nine to 10 mice bearing ISOS-1 angiosarcoma. All statistical tests were two-sided. We discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in endothelial cells, promoting statistically significant (P < .001) cell death when cells are confluent (mean [SD] % viability: control DLC1 = 15.6 [19.3]; shDLC1 = 73.4 [13.1]). This prosurvival phenotype of DLC1-depleted confluent endothelial cells is attributable to a statistically significant and sustained increase of NF-κB activity (day 5, P = .001; day 8, P = .03) associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistently, we found that DLC1 is statistically significantly reduced (P < .001 in 5 of 6) and TNFAIP3/A20 is statistically significantly increased (P < .001 in 2 of 3 and P = 0.02 in 1 of 3) in human angiosarcoma compared with normal adjacent endothelium. Treatment with the NF-κB inhibitor Tanespimycin/17-AAG statistically significantly reduced angiosarcoma tumor growth in mice (treatment tumor weight vs control, 0.50 [0.19] g vs 0.91 [0.21] g, P = .001 experiment 1; 0.66 [0.26] g vs 1.10 [0.31] g, P = .01 experiment 2). These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma and support NF-κB targeting for the treatment of angiosarcoma where DLC1 is lost.

Biallelic Dicer1 Loss Mediated by aP2-Cre Drives Angiosarcoma.

Angiosarcoma is an aggressive vascular sarcoma with an extremely poor prognosis. Because of the relative rarity of this disease, its molecular drivers and optimal treatment strategies are obscure. DICER1 is an RNase III endoribonuclease central to miRNA biogenesis, and germline DICER1 mutations result in a cancer predisposition syndrome, associated with an increased risk of many tumor types. Here, we show that biallelic Dicer1 deletion with aP2-Cre drives aggressive and metastatic angiosarcoma independent of other genetically engineered oncogenes or tumor suppressor loss. Angiosarcomas in aP2-Cre;Dicer1Flox/- mice histologically and genetically resemble human angiosarcoma. miR-23 target genes, including the oncogenes Ccnd1 as well as Adam19, Plau, and Wsb1 that promote invasiveness and metastasis, were enriched in mouse and human angiosarcoma. These studies illustrate that Dicer1 can function as a traditional loss-of-function tumor suppressor gene, and they provide a fully penetrant animal model for the study of angiosarcoma development and metastasis. Cancer Res; 77(22); 6109-18. ©2017 AACR.

Survivin: A novel marker and potential therapeutic target for human angiosarcoma.

Human angiosarcoma is a rare malignant vascular tumor associated with extremely poor clinical outcome and generally arising in skin of the head and neck region. However, little is known about the molecular pathogeneses and useful immunohistochemical markers of angiosarcoma. To investigate the mechanisms of angiosarcoma progression, we collected 85 cases of human angiosarcoma specimens with clinical records and analyzed ISO‐HAS‐B patient‐derived angiosarcoma cells. As control subjects, 54 cases of hemangioma and 34 of pyogenic granuloma were collected. Remarkably, consistent with our recent observations regarding the involvement of survivin expression following Hippo pathway inactivation in the neoplastic proliferation of murine hemangioendothelioma cells and human infantile hemangioma, nuclear survivin expression was observed in all cases of angiosarcoma but not in hemangiomas and pyogenic granulomas, and the Hippo pathway was inactivated in 90.3% of yes‐associated protein (YAP) ‐positive angiosarcoma cases. However, survivin expression modes and YAP localization (Hippo pathway activation modes) were not correlated with survival. In addition, we confirmed that survivin small interference RNA (siRNA) transfection and YM155, an anti‐survivin drug, elicited decreased nuclear survivin expression and cell proliferation in ISO‐HAS‐B cells which expressed survivin consistently. Conclusively, these findings support the importance of survivin as a good marker and critical regulator of cellular proliferation for human angiosarcoma and YM155 as a potential therapeutic agent.

MiR-497-5p inhibits cell proliferation and invasion by targeting KCa3.1 in angiosarcoma.

Angiosarcoma is a rare malignant mesenchymal tumor with poor prognosis. We aimed to identify malignancy-associated miRNAs and their target genes, and explore biological functions of miRNA and its target in angiosarcoma. By miRNA microarrays and reverse transcription polymerase chain reaction, we identified 1 up-regulated miRNA (miR-222-3p) and 3 down-regulated miRNAs (miR-497-5p, miR-378-3p and miR-483-5p) in human angiosarcomas compared with human capillary hemangiomas. The intermediate-conductance calcium activated potassium channel KCa3.1 was one of the putative target genes of miR-497-5p, and marked up-regulation of KCa3.1 was detected in angiosarcoma biopsy specimens by immunohistochemistry. The inverse correlation of miR-497-5p and KCa3.1 also was observed in the ISO-HAS angiosarcoma cell line at the mRNA and protein levels. The direct targeting of KCa3.1 by miR-497-5p was evidenced by reduced luciferase activity due to complementary binding of miR-497-5p to KCa3.1 mRNA 3′ untranslated region. For the functional role of miR-497-5p/KCa3.1 pair, we showed that application of TRAM-34, a specific KCa3.1 channel blocker, or transfection of ISO-HAS cells with KCa3.1 siRNA or miR-497-5p mimics inhibited cell proliferation, cell cycle progression, and invasion by down-regulating cell-cycle related proteins including cyclin D1, surviving and P53 and down-regulating matrix metallopeptidase 9. In an in vivo angiosarcoma xenograft model, TRAM-34 or miR-497-5p mimics both inhibited tumor growth. In conclusion, the tumor suppressor miR-497-5p down-regulates KCa3.1 expression and contributes to the inhibition of angiosarcoma malignancy development. The miR-497-5p or KCa3.1 might be potential new targets for angiosarcoma treatment.

Abstract LB-038: Constitutive activation of mTORC1 in endothelial cells leads to the development and progression of lymphangiosarcoma through VEGF autocrine signaling

Abstract Angiosarcoma/ lymphangiosarcoma is a rare malignancy with poor prognosis, for which animal models with a defined molecular pathogenesis are lacking. Here, we generated a new mouse model with an inducible endothelial cell specific deletion of Tsc1 to examine the potential role of abnormal activation of mTORC1 signaling in lymphangiosarcoma. We found that loss of Tsc1 increased EC proliferation and retinal angiogenesis in neonates. In adult mice, Tsc1 deletion led to a spectrum of endothelial proliferative lesions, ranging from vascular malformations to vascular tumors. Studies using rapamycin indicated that sustained mTORC1 signaling is required for both the development and maintenance of lymphangiosarcomas. Further analysis showed increased VEGF expression through increased activity of HIF1α and c-Myc transcription factors in tumor cells, and that blockade of autocrine VEGF signaling decreased proliferation of these cells both in vitro and in vivo. Lastly, we found significant correlations between mTORC1 activation and expression of VEGF, HIF1α and c-Myc in human angiosarcoma samples. Together, these studies demonstrate critical mechanisms of aberrant mTORC1 activation in the development of lymphangiosarcoma, and validate Tsc1iΔEC mice as a valuable novel model of lymphangiosarcoma with known molecular underpinnings for further study. Citation Format: Shaogang Sun, Song Chen, Fei Liu, Haige Wu, Jonathan McHugh, Ingrid Bergin, Anita Gupta, Denise Adams, Jun-Lin Guan. Constitutive activation of mTORC1 in endothelial cells leads to the development and progression of lymphangiosarcoma through VEGF autocrine signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-038.

Overexpression of Peroxiredoxin 6 Protects Neoplastic Cells against Apoptosis in Canine Haemangiosarcoma

Canine haemangiosarcoma (HSA), like human angiosarcoma, is an uncommon malignant vascular endothelial cell tumour associated with a poor prognosis. The peroxiredoxin (PRDX) family of peroxidases, which comprises six members in mammals (PRDX1-6), might contribute to cancer cell survival in the face of oxidative stress as these proteins exhibit frequent upregulation in cancer cells. In this study, we investigated the expression levels of PRDX6 in spontaneously arising primary canine HSAs by immunohistochemical analysis, identifying marked expression of this protein. Both PRDX6 mRNA and protein were overexpressed in HSA cell lines compared with normal canine endothelial cells, although some variation was observed between the different HSA cell lines. Small interfering RNA-induced downregulation of PRDX6 promoted apoptosis in the HSA cell lines. The observation that PRDX6 suppression increased the cytotoxicity of these cells suggests that PRDX6 might play an important cytoprotective role.

Prognostic Significance of Forkhead Box M1 (FOXM1) Expression and Antitumor Effect of FOXM1 Inhibition in Angiosarcoma.

Background: The prognosis of angiosarcoma is poor and a novel treatment option for the disease is desired. The aim of this study was to investigate the prognostic significance of Forkhead box M1 (FOXM1), a transcription factor that regulates cell-cycle progression and various crucial processes in tumor progression, and its potential as a new therapeutic target.Methods: We investigated 125 angiosarcoma clinical samples (94 primary lesions and 31 metastatic lesions in 94 patients) and a human angiosarcoma cell line (HAMON) using immunohistochemical staining and molecular biological approaches. FOXM1 expression in angiosarcoma samples was also compared with that in Kaposi's sarcomas (n = 13), epithelioid hemangioendotheliomas (n = 13) and benign hemangiomas (n = 10).Results: Patients with FOXM1-overexpressing angiosarcoma had significantly shorter survival (both for disease-specific survival [DSS] and event-free survival [EFS]) than other patients (5-year DSS, 23.5% vs. 47.1%, P = 0.013; and 5-year EFS, 5.5% vs. 28.7%, P = 0.004). FOXM1 overexpression was also an independent prognostic factor for both DSS and EFS in Cox multivariate analyses (hazard ratio [HR] 2.84, 95% confidence interval [CI] 1.10-5.81, P = 0.039; and HR 4.16, 95%CI 2.03-8.67, P = 0.0001, respectively). FOXM1 inhibition using both small interfering RNA and a specific inhibitor (thiostrepton) suppressed cell proliferation of the angiosarcoma cell line. Furthermore, FOXM1 inhibition improved the chemosensitivity to docetaxel in vitro.Conclusions: FOXM1 inhibition may be a potential therapeutic option for angiosarcoma.

Constitutive Activation of mTORC1 in Endothelial Cells Leads to the Development and Progression of Lymphangiosarcoma through VEGF Autocrine Signaling

Angiosarcoma/lymphangiosarcoma is a rare malignancy with poor prognosis. We generated a mouse model with inducible endothelial-cell-specific deletion of Tsc1 to examine mTORC1 signaling in lymphangiosarcoma. Tsc1 loss increased retinal angiogenesis in neonates and led to endothelial proliferative lesions from vascular malformations to vascular tumors in adult mice. Sustained mTORC1 signaling was required for lymphangiosarcoma development and maintenance. Increased VEGF expression in tumor cells was seen, and blocking autocrine VEGF signaling abolished vascular tumor development and growth. We also found significant correlations between mTORC1 activation and VEGF, HIF1α, and c-Myc expression in human angiosarcoma samples. These studies demonstrated critical mechanisms of aberrant mTORC1 activation in lymphangiosarcoma and validate the mice as a valuable model for further study.

NUP160-SLC43A3 is a novel recurrent fusion oncogene in angiosarcoma.

Angiosarcoma is a malignant vascular tumor originating from endothelial cells of blood vessels or lymphatic vessels. The specific driver mutations in angiosarcoma remain unknown. In this study, we investigated this issue by transcriptome sequencing of patient-derived angiosarcoma cells (ISO-HAS), identifying a novel fusion gene NUP160-SLC43A3 found to be expressed in 9 of 25 human angiosarcoma specimens that were examined. In tumors harboring the fusion gene, the duration between the onset of symptoms and the first hospital visit was significantly shorter, suggesting more rapid tumor progression. Stable expression of the fusion gene in nontransformed human dermal microvascular endothelial cells elicited a gene-expression pattern mimicking ISO-HAS cells and increased cell proliferation, an effect traced in part to NUP160 truncation. Conversely, RNAi-mediated attenuation of NUP160 in ISO-HAS cells decreased cell number. Confirming the oncogenic effects of the fusion protein, subcutaneous implantation of NUP160-SLC43A3-expressing fibroblasts induced tumors resembling human angiosarcoma. Collectively, our findings advance knowledge concerning the genetic causes of angiosarcoma, with potential implications for new diagnostic and therapeutic approaches.

MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases

Malignant endothelial proliferative diseases including human angiosarcoma (AS) and canine hemangiosarcoma (HSA) are serious diseases with a grave prognosis. Establishing liquid biopsy-based biomarkers for screening has definite clinical utility; however, plasma miRNAs up- or down-regulated in these sarcomas have been unclear. For identifying possible diagnostic plasma miRNAs for these sarcomas, we investigated whether plasma miR-214 and miR-126, which miRNAs play important roles in angiogenesis and tumorigenesis, were elevated in malignant endothelial proliferative diseases. For this investigation, human angiosarcoma and canine hemangiosarcoma cell lines and clinical plasma samples of canine hemangiosarcoma were examined by performing miRNA qRT-PCR. We report here that human angiosarcoma and canine hemangiosarcoma cell lines over-secreted miR-214 and miR-126 via microvesicles; in addition, their levels in the plasma samples from canines with hemangiosarcoma were increased. Moreover, the surgical resection of primary tumors decreased the levels of plasma miR-214 and miR-126. Our findings suggest that these malignant endothelial proliferative diseases over-secreted miR-214 and miR-126, thus suggesting that these miRNAs have potential as diagnostic biomarkers for malignant endothelial proliferative diseases in canine and possible in human angiosarcoma.

Beta Adrenergic Signaling: A Targetable Regulator of Angiosarcoma and Hemangiosarcoma.

Human angiosarcomas and canine hemangiosarcomas are highly aggressive cancers thought to arise from cells of vascular origin. The pathological features, morphological organization, and clinical behavior of canine hemangiosarcomas are virtually indistinct from those of human angiosarcomas. Overall survival with current standard-of-care approaches remains dismal for both humans and dogs, and each is likely to succumb to their disease within a short duration. While angiosarcomas in humans are extremely rare, limiting their study and treatment options, canine hemangiosarcomas occur frequently. Therefore, studies of these sarcomas in dogs can be used to advance treatment approaches for both patient groups. Emerging data suggest that angiosarcomas and hemangiosarcomas utilize beta adrenergic signaling to drive their progression by regulating the tumor cell niche and fine-tuning cellular responses within the tumor microenvironment. These discoveries indicate that inhibition of beta adrenergic signaling could serve as an Achilles heel for these tumors and emphasize the need to design therapeutic strategies that target tumor cell and stromal cell constituents. In this review, we summarize recent discoveries and present new hypotheses regarding the roles of beta adrenergic signaling in angiosarcomas and hemangiosarcomas. Because the use of beta adrenergic receptor antagonists is well established in human and veterinary medicine, beta blockade could provide an immediate adjunct therapy for treatment along with a tangible opportunity to improve upon the outcomes of both humans and dogs with these diseases.

MicroRNA-214 Promotes Apoptosis in Canine Hemangiosarcoma by Targeting the COP1-p53 Axis.

MicroRNA-214 regulates both angiogenic function in endothelial cells and apoptosis in various cancers. However, the regulation and function of miR-214 is unclear in canine hemangiosarcoma, which is a spontaneous model of human angiosarcoma. The expression and functional roles of miR-214 in canine hemangiosarcoma were presently explored by performing miRNA TaqMan qRT-PCR and transfecting cells with synthetic microRNA. Here, we report that miR-214 was significantly down-regulated in the cell lines used and in clinical samples of canine hemangiosarcoma. Restoration of miR-214 expression reduced cell growth and induced apoptosis in canine hemangiosarcoma cell lines through transcriptional activation of p53-regulated genes although miR-214 had a slight effect of growth inhibition on normal endothelial cells. We identified COP1, which is a critical negative regulator of p53, as a novel direct target of miR-214. COP1 was overexpressed and the specific COP1 knockdown induced apoptosis through transcriptional activation of p53-regulated genes as well as did miR-214-transfection in HSA cell lines. Furthermore, p53 knockdown abolished the miR-214-COP1-mediated apoptosis; thus, miR-214 and COP1 regulated apoptosis through controlling p53 in HSA. In conclusion, miR-214 functioned as a tumor suppressor in canine hemangiosarcoma by inducing apoptosis through recovering the function of p53. miR-214 down-regulation and COP1 overexpression is likely to contribute to tumorigenesis of HSA. Therefore, targeting miR-214-COP1-p53 axis would possibly be a novel effective strategy for treatment of canine hemangiosarcoma and capable of being applied to the development of novel therapeutics for human angiosarcoma.

Abstract 1694: Preclinical evaluation of a novel bispecific targeted toxin for the treatment of sarcomas

Abstract Introduction: EGFuPA is a bispecific ligand-targeted toxin consisting of epidermal growth factor (EGF) and urokinase (uPA) on the same molecule with truncated Pseudomonas exotoxin. The toxin was mutated to remove amino acids recognized by B-cells. EGFuPA is uniquely designed to simultaneously target EGF receptors (EGFR) and/or urokinase receptors (uPAR) on tumor cells and uPAR in the microenvironment. EGFuPA is safe and effective in laboratory animals and has excellent in vitro activity against sarcomas. Because EGFuPA crosses species platforms, an adaptive phase 1/2 study was undertaken using canine hemangiosarcoma (HSA) as a model of a highly aggressive, metastatic vascular sarcoma. We hypothesized that six-month survival would exceed 50% for dogs enrolled. Canine HSA is a tumor derived from blood vessel forming cells, which can overexpress both uPAR and EGFR rendering it an excellent target for the drug. It bears similarity to human angiosarcoma. Methods: Enrollment began in November 2012. Dogs with histologically confirmed, non-metastatic stage-I or II splenic HSA were eligible after splenectomy. EGFuPA was administered intravenously for one cycle on Days 1, 3, and 5. Adjuvant doxorubicin was given according to standard of care (SOC) protocols starting seven days after the last dose of EGFuPA. Results: EGFuPA was detectable in the systemic circulation 5 minutes post-infusion with clearance by 15 minutes. The drug was well tolerated with self-limiting, reversible hypovolemia at the highest dose. Thus far, 50 μg/kg appears to be the optimal dose. When compared to a historical control group of 19 dogs treated with SOC between 2008 and 2011, median survival is significantly longer for all 16 dogs treated with EGFuPA (SRCBST group, 240 vs. 129 days, p = 0.03) and for the 10 dogs in the Optimal Dose group (MS not reached, p = 0.01). Six-month survival for the Optimal Dose group was 65%. Overall survival at 400 days was 7% for the SOC group, 32.7% for the SRCBST group, and 56% for the Optimal Dose group. Outcomes were not affected by the presence of neutralizing antibodies. Conclusions: A biologically active dose of EGFuPA was established and found to be safe in companion dogs with HSA in the minimal residual disease setting. The unique nature of its bispecific targeting and the observed significant, biologic activity warrants consideration of EGFuPA for a phase 1 study for drug-refractory sarcomas in human patients. Citation Format: Antonella Borgatti, Amber Winter, Kathleen Stuebner, Joseph Koopmeiners, Jaime F. Modiano, Daniel Vallera. Preclinical evaluation of a novel bispecific targeted toxin for the treatment of sarcomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1694. doi:10.1158/1538-7445.AM2015-1694

Abstract 2092: FOXO1/Sprouty2 pathway regulates vascular tumor growth

Abstract Vascular tumors are endothelial cell neoplasms with a wide spectrum of clinical presentations, ranging from benign infantile hemangiomas in children to low-grade malignant hemangioendotheliomas and highly aggressive angiosarcomas in adults. To date, the molecular basis of vascular tumor pathogenesis is poorly understood and standard therapy for these tumors have limited clinical efficacy. Forkhead box protein O1 (FOXO1) is a transcription factor with tumor suppressor function and is dysregulated in human cancer. In this study, we showed that FOXO1 suppressed vascular tumor growth, and mechanistically, the inhibitory effects of FOXO1 were mediated by Sprouty2. FOXO1 expression was reduced in a variety of human vascular tumors examined (infantile hemangioma, hemangioendothelioma and angiosarcoma) as compared with normal blood vessels as determined by western blotting and immunohistochemical stains. Knockdown of FOXO1 gene expression with short hairpin RNA resulted in increased vascular tumor cell migration and proliferation in vitro and in vivo. Conversely, over-expression of constitutively active FOXO1 in these cells suppressed cell growth. We observed that FOXO1 interacted with Sprouty2 promoter in situ in chromatin immunoprecipitation assay and increased Sprouty2 gene expression in tumor cells. Similar to FOXO1, Sprouty2 expression was reduced in vascular tumors. Over-expression of Sprouty2 decreased tumor cell growth and migration. Conversely, knockdown of Sprouty2 increased tumor growth in vitro and in vivo. Knockdown of Sprouty2 in cells with over-expression of constitutively active FOXO1 resulted in reduced tumor growth and “rescued” the FOXO1 phenotype, indicating that Sprouty2 is an important mediator of the biological effects of FOXO1. Microarray gene expression profiling of human angiosarcoma cells with Sprouty2 knockdown together with network data integration using bioinformatics analysis and validated by quantitative PCR revealed important Sprouty2-regulated genes that are involved in angiogenesis, apoptosis and growth signal transduction pathways, including the collagen gene family, Notch signaling pathway and the GTPase IMAP family members. In summary, these findings demonstrate important growth regulatory role of the FOXO1/Sprouty2 pathway in vascular tumors and highlight the potential roles of novel pathways downstream of Sprouty2 in these tumors. Citation Format: Sriram Ayyaswamy, Wa Du, Christopher Anderson, Thuy L. Phung. FOXO1/Sprouty2 pathway regulates vascular tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2092. doi:10.1158/1538-7445.AM2015-2092

Genome-wide Association Study Identifies Shared Risk Loci Common to Two Malignancies in Golden Retrievers

Dogs, with their breed-determined limited genetic background, are great models of human disease including cancer. Canine B-cell lymphoma and hemangiosarcoma are both malignancies of the hematologic system that are clinically and histologically similar to human B-cell non-Hodgkin lymphoma and angiosarcoma, respectively. Golden retrievers in the US show significantly elevated lifetime risk for both B-cell lymphoma (6%) and hemangiosarcoma (20%). We conducted genome-wide association studies for hemangiosarcoma and B-cell lymphoma, identifying two shared predisposing loci. The two associated loci are located on chromosome 5, and together contribute ~20% of the risk of developing these cancers. Genome-wide p-values for the top SNP of each locus are 4.6×10-7 and 2.7×10-6, respectively. Whole genome resequencing of nine cases and controls followed by genotyping and detailed analysis identified three shared and one B-cell lymphoma specific risk haplotypes within the two loci, but no coding changes were associated with the risk haplotypes. Gene expression analysis of B-cell lymphoma tumors revealed that carrying the risk haplotypes at the first locus is associated with down-regulation of several nearby genes including the proximal gene TRPC6, a transient receptor Ca2+-channel involved in T-cell activation, among other functions. The shared risk haplotype in the second locus overlaps the vesicle transport and release gene STX8. Carrying the shared risk haplotype is associated with gene expression changes of 100 genes enriched for pathways involved in immune cell activation. Thus, the predisposing germ-line mutations in B-cell lymphoma and hemangiosarcoma appear to be regulatory, and affect pathways involved in T-cell mediated immune response in the tumor. This suggests that the interaction between the immune system and malignant cells plays a common role in the tumorigenesis of these relatively different cancers.

Human Angiosarcoma: A Histological and Biological Phenotyping Using Xenografts in Nude Mice: Analysis of Five Cases

Human angiosarcoma (AS) is an infrequent soft tissue malignancy that may be difficult to diagnose, especially the solid, epithelioid or pleomorphic variant. A broad panel of antibodies is often necessary to determine the phenotype by Immunohistochemistry (IHC). To improve our knowledge of the morphology, immunophenotype, genetics and molecular biology, we constructed an in vivo xenograft platform using human soft tissue AS in nude mice. Tissue Microarrays (TMAs) were built from the original tumors and subsequent passages. The study included four original human AS, plus one secondary (a primary Malignant Peripheral Nerve Sheath Tumor (MPNST) which became an angiosarcoma), followed over successive tumor transfers for several generations. Endothelial marker expression (CD31, CD34, FVIII, CD105 (endoglin), Caveolin (CAV-1), D2-40, V-cadherin, FLI1 and ERG n-terminal) was evaluated by IHC. Molecular and cytogenetics studies were also performed. Only three out of the five AS (60%), grew in nude mice. Between 30 and 52 passages were achieved. Cytogenetically, the tumors showed complex karyotypes with many clonal numerical and structural abnormalities. Molecular biology showed p53 wild-type and KDR in all the cases. The WWTR1-CAMTA fusion was absent, which may be helpful in distinguishing epithelioid AS from epithelioid hemangioendothelioma (EHE), as it has been described as a hallmark in the latter. CD31, ERG, D2-40 and V-cadherin were the most sensitive, and ERG and FLI1 the most specific markers in the present series. This is the first study to combine nude mice xenografts and TMA studies in human AS, and describes for the first time an angiosarcomatous differentiation of an MPNST which occurred during the late passages generating a Kasabach-Merritt-like syndrome in the animal.

Angiogenic growth factor expression in benign and malignant vascular tumours

Angiosarcomas are rare malignant vascular tumours. Angiosarcoma expression of vascular endothelial growth factor (VEGF) has previously been reported, but angiosarcoma expression of other angiogenic growth factors has not been systematically studied. Non-VEGF angiogenic growth factors are a potential mechanism of resistance to VEGF-targeted therapy, but they also represent potential therapeutic targets.Immunohistochemistry analysis evaluated the expression of 13 angiogenic growth factors and receptors in 27 separate benign and malignant archived human vascular tumour samples. The expression of 55 angiogenesis-related proteins was subsequently profiled in five fresh human angiosarcoma tumour samples using antibody arrays.Angiosarcomas expressed a variety of angiogenic growth factors. Significantly higher levels of Notch1 were detected compared with benign haemangiomas (p=0.033), but lower levels of basic fibroblast growth factor (bFGF) compared to benign haemangiomas (p=0.07) and inflammatory vascular lesions (p=0.009). Vascular tumour expression of FGF receptor (FGFR)-1 correlated with angiopoietin (Ang)-2, Tie2, hepatocyte growth factor (HGF) and Notch1 expression (p=0.001, p=0.001, p<0.001 and p<0.001 respectively). Notch1 also correlated with Tie2 expression (p=0.004).In conclusion, angiosarcomas express multiple angiogenic growth factors. Treatments could be targeted at individual angiogenic growth factors. However, our findings provide a rationale for combination therapy, or for treatments that target common downstream signalling intermediaries, such as Akt, mTOR or ERK.