Abstract 386: Breaking the harmony of TNF-α signaling for cancer treatment
Authors: Sasi Shararth P. , Park Daniel , Hlatky Lynn , Gilbert Hu-Ya , Hahnfeldt Philip , Kunder Sharon , Coleman Christina , Qin Qangjian , Kishore Raj , Goukassian David A. 
Published: June 2012

Abstract
Tumor necrosis factor-alpha(TNF) binds two distinct receptors TNFR1/p55 and TNFR2/p75 and is implicated in processes of tumor growth, survival, differentiation, invasion, metastases, secretion of cytokines and pro-angiogenic factors. We hypothesized that inhibition of signaling via one or the other but not both TNF receptors may have deleterious effect on tumor growth.
We injected WT, p55 knockout(KO), p75KO and double p55KO/p75KO mice with 5×105 mouse Lewis lung carcinoma line (LLC1) mixed with Matrigel into the flank. Compared to day 1, tumors in WTs and p55KO/p75KOs became ∼343 % and ∼ 933% larger between days 14 and 21, respectively, whereas at the same period, tumor growth was inhibited in both p75KOs (46% and 51%, days 14 and 21, p&lt;0.05) and p55KOs (42% and 38%, days 14 and 21, p&lt;0.05). Tumors bisected from all genotypes 14 days post-inoculation were collected and processed for immunostaining for CD31, TUNEL, TNF and VEGF expression and imaged by confocal microscopy. TNF levels were significantly higher within genotype in tumor tissue vs normal skin in WT 10-fold (p&lt;0.001), in p75KO 8.7-fold (p&lt;0.001) and p55KO 6-fold (p&lt;0.03). Compared to WTs, CD31 immunostaining revealed 80% (p&lt;0.001) and 60% (p&lt;0.02) decreases in capillary density in tumors from p75KO and p55KO, respectively. TUNEL staining showed ∼50% increases in TUNEL (+) cells in tumors of both p75KO and p55KO. VEGF expression was decreased more than 50% (p&lt;0.001) in p75KO and 40% (p&lt;0.02) in p55KO. Bone marrow(BM)-derived endothelial progenitor cells(EPC) were tracked into the tumor using BM/GFP transplantation model in combination with BS1-lectin perfusion and staining. Compared to WT and p55KOs there was ∼50% (p&lt;0.05) decrease of BM-derived EPCs incorporation into the functional capillary network in p75KOs. Alterations in angiogenic and apoptotic signaling pathways in whole tumor tissue were analyzed using gene array analysis, which revealed that absence of either receptor had significant inhibitory effect (&gt;2-fold) on several genes of angiogenic, anti-apoptotic and pro-survival pathways that include, but not limited to, VEGF A and B, HIF1-alpha, MAPK14(p38), HGF, IL1b, IL12a, Pgf, NFkB, Faim, Api5, Bnip3, Cxcl1, 2 and 10.
Our results indicate that absence of the signaling through either p75 or p55 in the tumor microenvironment only inhibits tumor growth by ∼50% by negatively regulating tumor angiogenesis, suggesting that inhibition of p75 or p55 in tumor tissue in vivo may deliver “double hit” by affecting survival of ECs, while higher TNF levels in tumor tissue may have self-destructive effect. Significantly higher deficiency in tumor angiogenesis in p75KOs vs p55KOs demonstrate inability of host p75KO ECs to survive in “hostile” tumor microenvironment (high TNF) as well as impede incorporation of BM-derived p75KO EPC's into tumor vasculature, suggesting that inhibition of tumor angiogenesis is a primary mechanism of tumor growth inhibition in p75KOs.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 386. doi:10.1158/1538-7445.AM2011-386