Abstract 676: Tissue inhibitor of metalloproteinase-2 (TIMP2) deficiency enhances tumor burden via increasing HIF-2á expression

Abstract

Abstract The tissue inhibitor of metalloproteinase family of proteins (TIMPs 1-4) function as natural MMP inhibitors, and have been shown to play a role in maintenance and remodeling of the ECM as well as other cellular processes including proliferation, apoptosis and angiogenesis. A number of studies have shown that the down-regulation or silencing of TIMP2 accelerates tumor development, however, the mechanism is not well understood. High HIF-2á levels in non-small cell lung cancer (NSCLC) correlate with decreased overall survival, while inhibition of HIFs targeted genes VEGF or VEGFR2 are associated with improved clinical outcome. Similarly, TIMP2 mRNA levels were found to be low in NSCLC compared to the corresponding non-neoplastic surrounding lung (p<0.05). The current study was undertaken to examine the molecular mechanisms associated with tumor hypoxia and TIMP2 expression in TIMP2 deficient mice (T2D-/-) and wild type (WT) littermates. Mice were given (1×106)/50ìl Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) via intratracheal installation. Tumor progression was monitored by IVIS imaging 2 weeks after administration and continued until week 4 when mice were sacrificed and lungs harvested for further analysis. The IVIS analysis shows higher tumor burden in T2D-/- compared to WT littermates, suggesting that TIMP2 deficiency augments tumor development in these mice (p<0.05). H&E analysis of lung tissue sections revealed a significant increase in the number of tumor nodules in lungs of T2D mice compared to controls (p = 0.003). In an effort to investigate the relationship between TIMP2 and HIF-2á expression; we determined the expression levels of HIF-2á in healthy non-tumor bearing T2D mice compared to WT littermates. Using semi-quantitative real-time PCR, we confirmed a strong basal up regulation of HIF-2á (but not HIF-1á) mRNA in T2D mice lungs compared to WT (p<0.002). In tumor bearing mice, this increase was even higher in the lungs of T2D mice compared to WT (p<0.007). Since VEGF is the direct downstream target of HIF-2á, we determined the expression of VEGF in these mice. As expected, we found a similar trend of VEGF expression in T2D mice. The basal level of VEGF expression was increased in the lungs of healthy T2D mice compared to WT (p<0.006) and even higher in tumors of T2D mice compared WT (p<0.002). Given that the HIF-2á−VEGF signaling pathway plays a major role in neovascularization and cancer progression, microvessel density was assessed in tumor tissue by calculating the mean number of CD31+ vessels. Accordingly, we found a higher number of CD31+ vessels in T2D mice compared to WT (p<0.0001). Collectively, these findings support our previous work on demonstrating TIMP2 as an inhibitor of tumor angiogenesis and also suggest that TIMP2 regulates hypoxic mediators within the tumor microenvironment, therefore offering TIMP2 as a novel bio-therapeutic molecule for lung cancer therapy. Citation Format: Sarvesh Kumar, Sandra M. Jensen, Ananda Chowdhury, Beiyang Wei, William G. Stetler-Stevenson. Tissue inhibitor of metalloproteinase-2 (TIMP2) deficiency enhances tumor burden via increasing HIF-2á expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 676.