Abstract 168: Loss of function mutation in TIMP2 gene accelerates tumorigenesis and mortality in murine model of lung cancer through EGFR signaling
Authors: Kumar Sarvesh , Jensen Sandra , Peeney David , Chowdhury Ananda , Wei Beiyang , Stetler-Stevenson William G. 
Published: July 2017

The aim of this study is to determine the effect of the tissue inhibitor of metalloproteinase-2 (TIMP-2) on lung tumorigenesis and investigate the underlying molecular mechanisms using an orthotopic mouse model with a loss of function mutation in the Timp2 gene (T2M). T2M and wildtype (WT) control mice were given 1x106 Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) in 50μL PBS via intratracheal installation. IVIS imaging revealed a higher tumor burden in T2M mice compared to WT littermates, suggesting that loss of function of the Timp2 gene enhances tumor growth (p&lt;0.05). We also conducted a Kaplan-Meier analysis to determine the effect of this mutation on mortality following cancer development. We found that LL/2 tumor-induced mortality was significantly higher in T2M mice compared to WT controls (p = 0.01). Histologic analysis and H&E staining of lung tissue sections revealed a significant increase in the number of tumor nodules of T2M mice compared to WT controls (p &lt;0.01). CD31 staining showed a significant increase in microvessel density (MVD) in T2M mice compared to WT controls (p&lt;0.01). Given that VEGF is a primary driver of tumor neovascularization, we determined mRNA levels of VEGF expression in healthy and tumor bearing mice by qRTPCR analysis. Interestingly, basal VEGF expression levels were increased in lungs of both non-tumor bearing and tumor bearing T2M mice compared to WT controls (p&lt;0.01). VEGF is the direct downstream target of HIF-2α, a transcription factor implicated in tumor hypoxia and expression correlates with decreased overall survival in non-small cell lung cancer (NSCLC) patients. Similarly, an examination of TIMP-2 and HIF-2α mRNA expression in a small cohort of patient samples revealed decreased TIMP-2 and increased HIF2α in NSCLC tumors compared to surrounding non-tumor lung tissue. Hypoxia induces EGFR signaling through HIF-2α. Thus, we determined the effect of mutated T2M on the EGFR signaling pathway. We found increased levels of EGFR phosphorylation as well as downstream ERK and Akt activation in tumor bearing and non-tumor bearing T2M lungs compared to WT controls. In conclusion, these findings offer new avenues for TIMP-2 research in regulating hypoxic mediators within the tumor microenvironment through EGFR signaling, suggesting TIMP-2 as a novel bio-therapeutic for lung cancer therapy.

* Corresponding author This work is supported by center for cancer research, intramural research program, NCI/NIH research grants ZIA BC011204 & ZIA SC 009179 to WGSS

Citation Format: Sarvesh Kumar, Sandra Jensen, David Peeney, Ananda Chowdhury, Beiyang Wei, William G. Stetler-Stevenson. Loss of function mutation in TIMP2 gene accelerates tumorigenesis and mortality in murine model of lung cancer through EGFR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 168. doi:10.1158/1538-7445.AM2017-168