ASA404 and TNF treatment in preclinical models of endocrine tumors is associated with differential TNF-R 1 and TLR- 4 signaling

Purpose: ASA404 (Vadimezan) belongs to a class of agents with disrupting properties against tumor vasculature, which is partly mediated by TNF-signaling. Our aim was to investigate the potential therapeutic applicability of ASA404 against endocrine tumors. Experimental Design: We determined anti-tumoral effects in preclinical models of neuroendocrine tumors of the gastroenteropancreatic system [1] and adrenocortical cancer (NCI-H295R) by histology and immunohistochemistry. Furthermore, we characterized these models with their clearly different responsiveness regarding TNF synthesis and signaling. Results: Upon treatment of tumor bearing mice significant anti-tumoral effects, an increase in TNF as well as TNF-specific activation of downstream signaling were evident in the BON tumor model while no comparable effects were detectable for NCI-H295R. Two important modulator of TNF-signaling, toll-like-receptor 4 (TLR-4) and its adaptor protein LY96 were found highly expressed in BON tumors and transgenic expression in NCI-H295R partly restored TNF responsiveness. Furthermore, expression of IRAK2-kinase, which has recently been linked to TLR-4-signaling as a mediator of sustained TNF release was induced upon TNF-treatment in BON, but not in NCI-H295R cells. Finally, we identified TNFAIP3/A20, a member of an inhibitory feedback-loop downstream of both investigated signaling cascades, as overexpressed in the adrenocortical carcinoma tumor model. Subsequent analyses of clinical patient samples confirmed a correlation between tumor TNFAIP3 expression levels and overall survival in patients with ACC. Conclusions: Taken together our findings provide evidence that modulation of TNF-signaling could be of relevance both for the clinical course of ACC patients and as a marker of treatment response.

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