Abstract 2317: Direct anti-angiogenic activity of a small molecular STAT3 inhibitor LLL12

Increased vascularization (angiogenesis) is a required adaptation for sustained tumor growth. It is a complex multistage process regulated by a number of signal transduction pathways, although the primary mediator of blood vessel formation is vascular endothelial growth factor (VEGF). Recent data indicate the Signal Transducer and Activator of Transcription 3 (STAT3) is required for VEGF production and angiogenesis in various types of cancers, possibly through regulating hypoxia inducible factor 1 (HIF-1). Other STAT3 inhibitors have been shown to reduce tumor microvessel density in tumors (Lin L et al., Cancer Res. 2010;70:2445-54), but a direct anti-angiogenic activity has not been described. In this study, we investigated the direct action of LLL12 in human umbilical chord vascular endothelial cells (HUVECs). LLL12 (100nM) significantly inhibited VEGF-stimulated STAT3 phosphorylation in HUVEC cells, reduced their proliferation/migration and also inhibited VEGF-induced tube formation. Morphologic analysis of LLL12 treated HUVECS demonstrated significant changes in actin/tubulin distribution and bundling. To test the anti-angiogenic effects of LLL12 in vivo, VEGF-infused Matrigel plugs were implanted into scid mice, and microvessel invasion (CD34 positive cells) determined after 7 days in vehicle treated or LLL12-treated mice. VEGF stimulated microvessel development (∼10-fold) over control plugs (PBS infused). The decreased in microvessel density in LLL12 treated mice was dose dependent, being reduced by ∼90% at a daily dose of 5 mg/kg. To test the antitumor activity of LLL12, mice bearing established OS-1 osteosarcoma xenografts with no treatment, vehicle alone or LLL12 (5 mg/kg daily) for 6 weeks. Following a period of tumor progression, LLL12 completely suppressed further growth. Pharmacodynamic studies at the end of treatment showed robust phosphor-STAT3 in control tumors whereas phosphor-STAT3 was not detected in treated OS-1 tumors. Treated tumors demonstrated decreased proliferation (Ki67 staining) but no significant increase in apoptosis (TUNEL staining), relative to controls. To examine the effect of LLL12 on angiogenic factors a Proteome Profiler antibody array was used to detect the relative levels of expression of 55 angiogenesis related proteins in control and treated OS-1 tumors. Several critical regulators of angiogenesis including VEGF, MMP-9, Angiopoieion1/2, Tissue Factor and FGF-1 expression were significantly reduced in LLL12 treated tumors compared to control OS-1 xenografts. These findings provide the first evidence that LLL12 effectively inhibits tumor angiogenesis both in vitro and in vivo. These data indicate that the inhibition of STAT3 signaling is a potential therapeutic approach for tumor angiogenesis, and tumor control. Supported in part through a Pelotonia grant from OSU. 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 2317. doi:1538-7445.AM2012-2317