Debarshi Banerjee

Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis

BackgroundAnti-angiogenesis is a validated strategy to treat cancer, with efficacy in controlling both primary tumor growth and metastasis. The role of the Notch family of proteins in tumor angiogenesis is still emerging, but recent data suggest that Notch signaling may function in the physiologic response to loss of VEGF signaling, and thus participate in tumor adaptation to VEGF inhibitors.MethodsWe asked whether combining Notch and VEGF blockade would enhance suppression of tumor angiogenesis and growth, using the NGP neuroblastoma model. NGP tumors were engineered to express a Notch1 decoy construct, which restricts Notch signaling, and then treated with either the anti-VEGF antibody bevacizumab or vehicle.ResultsCombining Notch and VEGF blockade led to blood vessel regression, increasing endothelial cell apoptosis and disrupting pericyte coverage of endothelial cells. Combined Notch and VEGF blockade did not affect tumor weight, but did additively reduce tumor viability.ConclusionsOur results indicate that Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis, and show that concurrent blockade disrupts primary tumor vasculature and viability further than inhibition of either pathway alone.

HAUSP deubiquitinates and stabilizes N-Myc in neuroblastoma

The MYCN proto-oncogene is amplified in a number of advanced-stage human tumors, such as neuroblastomas. Similar to other members of the MYC family of oncoproteins, MYCN (also known as N-Myc) is a transcription factor, and its stability and activity are tightly controlled by ubiquitination-dependent proteasome degradation. Although numerous studies have demonstrated that N-Myc is a driver of neuroblastoma tumorigenesis, therapies that directly suppress N-Myc activity in human tumors are limited. Here we have identified ubiquitin-specific protease 7 (USP7; also known as HAUSP) as a regulator of N-Myc function in neuroblastoma. HAUSP interacts with N-Myc, and HAUSP expression induces deubiquitination and subsequent stabilization of N-Myc. Conversely, RNA interference (RNAi)-mediated knockdown of USP7 in neuroblastoma cancer cell lines, or genetic ablation of Usp7 in the mouse brain, destabilizes N-Myc, which leads to inhibition of N-Myc function. Notably, HAUSP is more abundant in patients with neuroblastoma who have poorer prognosis, and HAUSP expression substantially correlates with N-Myc transcriptional activity. Furthermore, small-molecule inhibitors of HAUSPs deubiquitinase activity markedly suppress the growth of MYCN-amplified human neuroblastoma cell lines in xenograft mouse models. Taken together, our findings demonstrate a crucial role of HAUSP in regulating N-Myc function in vivo and suggest that HAUSP inhibition is a potential therapy for MYCN-amplified tumors.

Notch Suppresses Angiogenesis and Progression of Hepatic Metastases

The Notch pathway plays multiple key roles in tumorigenesis, and its signaling components have therefore aroused great interest as targets for emerging therapies. Here, we show that inhibition of Notch, using a soluble receptor Notch1 decoy, unexpectedly caused a remarkable increase in liver metastases from neuroblastoma and breast cancer cells. Increased liver metastases were also seen after treatment with the γ-secretase inhibitor PF-03084014. Transgenic mice with heterozygous loss of Notch1 demonstrated a marked increase in hepatic metastases, indicating that Notch1 signaling acts as metastatic suppressor in the liver microenvironment. Inhibition of DLL1/4 with ligand-specific Notch1 decoys increased sprouting of sinusoidal endothelial cells into micrometastases, thereby supporting early metastatic angiogenic growth. Inhibition of tumor-derived JAG1 signaling activated hepatic stellate cells, increasing their recruitment to vasculature of micrometastases, thereby supporting progression to macrometastases. These results demonstrate that inhibition of Notch causes pathologic activation of liver stromal cells, promoting angiogenesis and growth of hepatic metastases. Our findings have potentially serious implications for Notch inhibition therapy.

High-Dose, Single-Fraction Irradiation Rapidly Reduces Tumor Vasculature and Perfusion in a Xenograft Model of Neuroblastoma

PURPOSE To characterize the effects of high-dose radiation therapy (HDRT) on neuroblastoma tumor vasculature, including the endothelial cell (EC)-pericyte interaction as a potential target for combined treatment with antiangiogenic agents. METHODS AND MATERIALS The vascular effects of radiation therapy were examined in a xenograft model of high-risk neuroblastoma. In vivo 3-dimensional contrast-enhanced ultrasonography (3D-CEUS) imaging and immunohistochemistry (IHC) were performed. RESULTS HDRT significantly reduced tumor blood volume 6 hours after irradiation compared with the lower doses used in conventionally fractionated radiation. There was a 63% decrease in tumor blood volume after 12-Gy radiation compared with a 24% decrease after 2 Gy. Analysis of tumor vasculature by lectin angiography showed a significant loss of small vessel ends at 6 hours. IHC revealed a significant loss of ECs at 6 and 72 hours after HDRT, with an accompanying loss of immature and mature pericytes at 72 hours. CONCLUSIONS HDRT affects tumor vasculature in a manner not observed at lower doses. The main observation was an early reduction in tumor perfusion resulting from a reduction of small vessel ends with a corresponding loss of endothelial cells and pericytes.

Transcription factor activating protein 4 is synthetically lethal and a master regulator of MYCN- amplified neuroblastoma

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, MYCN inhibitors have yet to be developed. Here, by integrating evidence from a whole-genome shRNA library screen and the computational inference of master regulator proteins, we identify transcription factor activating protein 4 (TFAP4) as a critical effector of MYCN amplification in neuroblastoma, providing a novel synthetic lethal target. We demonstrate that TFAP4 is a direct target of MYCN in neuroblastoma cells, and that its expression and activity strongly negatively correlate with neuroblastoma patient survival. Silencing TFAP4 selectively inhibits MYCN-amplified neuroblastoma cell growth both in vitro and in vivo, in xenograft mouse models. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as evidenced by increased neurite outgrowth and upregulation of neuronal markers. Taken together, our results demonstrate that TFAP4 is a key regulator of MYCN-amplified neuroblastoma and may represent a valuable novel therapeutic target.

Abstract 2809: Deficiency of Notch-1 signaling in liver increases neuroblastoma hepatic metastases.

Purpose: The Notch signaling pathway has been extensively studied for its role in tumor angiogenesis but its function in tumor metastasis is largely elusive. In the present study we investigated the role of a notch receptor, Notch-1, in the regulation of neuroblastoma liver metastatic process. Methods: To investigate the function of Notch-1 in neuroblastoma liver metastasis we first generated immunodeficient (rag2gammaC deletion) Notch-1 heterozygous (+/-) mice. We crossed a Notch1 heterozygous (+/-) mouse with a rag2/gamma-c double knockout (-/-) mouse. The mouse with Notch1(+/-)rag2(+/-)gammaC(+/-) genotype was then backcrossed with a rag2gammaC double knockout (-/-) mouse to generate rag2(-/-)gammaC(-/-)Notch1(+/-) mouse or rag2(-/-)gammaC(-/-)Notch1(+/+)control mouse . These mice (n=5) were intracardically injected with 105 neuroblastoma NGP (MYCN amplified) cells expressing Firefly luciferase. The mice were subjected to IVIS- bioluminescence imaging, once in a week, to monitor the metastatic spread. All mice were sacrificed at week 8. At the time of sacrifice, mice were injected with Luciferin, sacrificed and liver was then dissected out, imaged and bioluminescence flux was measured (ex vivo imaging). The liver tissues were also sectioned (5um, paraffin) and HE 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2809. doi:10.1158/1538-7445.AM2013-2809

Abstract 2325: Increase in neuroblastoma metastasis after dual inhibition of VEGF and Notch

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: Vascular endothelial growth factor (VEGF) inhibition is a validated cancer treatment. However, even in responsive tumors, acquired resistance is common. Notch proteins also function as key angiogenic effectors, and cross-regulate VEGF expression, raising the question of whether combined treatment would enhance tumor suppression. We hypothesized that dual VEGF/Notch blockade would inhibit tumor growth in experimental SY5Y neuroblastoma. Methods: SY5Y neuroblastoma cells were lentivirally transfected to express Notch1-decoy (N1D) or GFP (control). Proliferation was assessed in vitro under both hypoxia and normoxia using BrdU assays. To examine the effect of N1D on tumor growth, 10[6] cells were xenografted intrarenally in nude mice and treated with placebo or bevacizumab (BV) twice a week. Tumor progression was monitored by bioluminescence. Metastatic burden in target organs was quantified by bioluminescence and histology. Vascular disruption due to N1D and BV treatment was evaluated by immunostaining. IACUC approval was obtained for all experiments. Results: Expression of soluble N1D was confirmed by immunoblotting conditioned media. SY5Y+N1D cells proliferated more rapidly in hypoxia than control SY5Y+GFP cells (p<0.01). In vivo, BV treatment alone significantly reduced tumor growth as compared to placebo (p=0.026). However, combined treatment (N1D + BV) did not further reduce SY5Y tumor growth as compared to BV treatment alone (p=0.684). Interestingly, dual-treated mice developed higher metastatic burdens in liver than mice treated with either agent alone or controls by bioluminescence (p=0.006). By histology, dual-treated mice displayed a higher incidence of liver metastasis (7/9 mice) vs. mice treated with BV only (3/10 mice). Immunostaining demonstrated disruption of tumor vessel architecture in BV- and N1D + BV-treated mice. Conclusion: Dual VEGF/Notch targeting of SY5Y tumors resulted in increased metastatic burden, without affecting primary tumor growth, as compared to either treatment alone. Recent data indicates that tumoral hypoperfusion can promote progression, potentially by selecting for biologically aggressive behaviors. Our results suggest that dual Notch/VEGF blockade causes an enhanced propensity to metastasize. These data warrant further preclinical investigation. 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 2325. doi:1538-7445.AM2012-2325