Radiotherapy followed by aurora kinase inhibition targets tumor-propagating cells in human glioblastoma.

Glioblastoma (GBM) is the most common malignant primary brain tumor. Radiotherapy fails to eliminate subpopulations of stem-like tumor-propagating cells (TPC), resulting in tumor regrowth. To identify kinases that promote TPC self-renewal rather than increasing proliferation in human GBM cultures, we screened a library of 54 nonselective tool compounds and determined their kinase inhibitor profiles in vitro. Most compounds inhibited aurora kinase (AURK) activity and blocked TPC self-renewal, while inducing GBM cell polynucleation and apoptosis. To prevent regrowth by TPCs, we used a priming dose of radiation followed by incubation with the pan-AURK inhibitor VX680 to block self-renewal and induce apoptosis in GBM cultures. In mice xenografted with human GBM cells, radiotherapy followed by VX680 treatment resulted in reduced tumor growth and increased survival relative to either monotherapy alone or VX680 treatment before radiation. Our results indicate that AURK inhibition, subsequent to radiation, may enhance the efficacy of radiotherapy by targeting radioresistant TPCs in human GBMs.

Targeting MYCN in neuroblastoma by BET bromodomain inhibition.

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.

Paracrine signaling through MYCN enhances tumor-vascular interactions in neuroblastoma.

Neuroblastoma, a tumor of peripheral neural crest origin, numbers among the most common childhood cancers. Both amplification of the proto-oncogene MYCN and increased neoangiogenesis mark high-risk disease. Because angiogenesis is regulated by phosphatidylinositol 3-kinase (PI3K), we tested a clinical PI3K inhibitor, NVP-BEZ235, in MYCN-dependent neuroblastoma. NVP-BEZ235 decreased angiogenesis and improved survival in both primary human (highly pretreated recurrent MYCN-amplified orthotopic xenograft) and transgenic mouse models for MYCN-driven neuroblastoma. Using both gain- and loss-of-function approaches, we demonstrated that the antiangiogenic efficacy of NVP-BEZ235 depended critically on MYCN in vitro and in vivo. Thus, clinical PI3K/mammalian target of rapamycin inhibitors drove degradation of MYCN in tumor cells, with secondary paracrine blockade of angiogenesis. Our data demonstrated significantly improved survival in treated animals and suggest that NVP-BEZ235 should be tested in children with high-risk, MYCN-amplified neuroblastoma.

Antiangiogenic therapy decreases integrin expression in normalized tumor blood vessels.

Tumor blood vessels normalized by antiangiogenic therapy may provide improved delivery of chemotherapeutic agents during a window of time but it is unknown how protein expression in tumor vascular endothelial cells changes. We evaluated the distribution of RGD-4C phage, which binds alpha(v)beta(3), alpha(v)beta(5), and alpha(5)beta(1) integrins on tumor blood vessels before and after antiangiogenic therapy. Unlike the control phage, fd-tet, RGD-4C phage homed to vascular endothelial cells in spontaneous tumors in RIP-Tag2 transgenic mice in a dose-dependent fashion. The distribution of phage was similar to alpha(v)beta(3) and alpha(5)beta(1) integrin expression. Blood vessels that survived treatment with AG-013736, a small molecule inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptors, had only 4% as much binding of RGD-4C phage compared with vessels in untreated tumors. Cellular distribution of RGD-4C phage in surviving tumor vessels matched the alpha(5)beta(1) integrin expression. The reduction in integrin expression on tumor vessels after antiangiogenic therapy raises the possibility that integrin-targeted delivery of diagnostics or therapeutics may be compromised. Efficacious delivery of drugs may benefit from identification by in vivo phage display of targeting peptides that bind to tumor blood vessels normalized by antiangiogenic agents.

Angiogenesis with pericyte abnormalities in a transgenic model of prostate carcinoma.

BACKGROUND: Previous studies of the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model vasculature suggest that, as tumors develop, vessels invade the glandular epithelium. However, changes in the vasculature are difficult to study in conventional thin tissue sections. The authors used a new approach to characterize morphologic and architectural changes of blood vessels and pericytes during tumor development in TRAMP mice. METHODS: Eighty-micron cryostat sections of normal prostate and three histopathologic stages of TRAMP tumor sections, classified by epithelial cell E-cadherin immunoreactivity, were immunostained with vascular endothelial cell and pericyte receptor antibodies and evaluated by confocal microscopy. RESULTS: In the normal mouse prostate, capillaries were most abundant in the fibromuscular tunica between the epithelium and smooth muscle of the ductules. In the prostatic intraepithelial neoplasia (PIN) stage, vessels accompanied epithelial cell protrusions into the ductule lumen but remained in the connective tissue at the basal side of the epithelium. Well differentiated tissues had extensive angiogenesis with five times the normal mean vascularity outside ductules. Vessels were of variable diameter, were associated with an increased number of pericytes, and some had endothelial sprouts. Angiogenic blood vessels from poorly differentiated adenocarcinomas were tortuous, variable in caliber, and lacked the normal hierarchy. Pericytes on these vessels had an abnormal phenotype manifested by alpha-smooth muscle actin expression and loose association with endothelial cells. Angiogenesis and loss of vascular hierarchy were also found in human prostate carcinoma. CONCLUSIONS: Vascular abnormalities, which begin at the PIN stage and intensify in well differentiated and poorly differentiated tumors, may be useful readouts for early detection and treatment assessment in prostate carcinoma.