The Role of Timp3 in the Pathogenesis of Colorectal Cancer and Timp3 Promoter Methylation as a Potential Predictive Marker for Egfr Inhibitor Therapy

Abstract

Introduction: Colorectal cancer (CRC) is a leading cause of cancer deaths in the United States. Current use of tumor stage to determine patient response to treatment is suboptimal. There is an underlying heterogeneity among the clinical behavior of CRC that likely reflects the molecular heterogeneity. Given the known toxicity of most chemotherapy, there is a critical need for biomarkers that can predict responsiveness to therapy. It is known that in approximately 30% of CRC, the tumor inhibitor of metalloproteinase-3 (TIMP3) promoter is hypermethylated, which is predicted to silence the expression of TIMP3. TIMP3 inhibits a disintegrin and metalloprotease- 17 (ADAM17), a matrix metalloproteinase responsible for ectodomain shedding of cell surface molecules that activate the epidermal growth factor receptor (EGFR) signaling pathway, as well as cleavage of cell surface receptors that promote apoptosis such as TNFR1. Increased ADAM17 activity results in increased shedding of amphiregulin, an EGFR ligand, and EGFR pathway activation. It is known that wild type KRAS tumors with high levels of amphiregulin are more responsive to the EGFR neutralizing monoclonal antibody, cetuximab. the purpose of this study is to determine if aberrant methylation of the TIMP3 promoter silences TIMP3 expression in CRC leading to increased amphiregulin and TNFR1 shedding. the results could provide a mechanism for increased EGFR pathway activation with simultaneous inhibition of apoptosis giving strong rationale for using the methylation status of the TIMP3 promoter as a predictive marker for responsiveness to chemotherapy with EGFR antagonists. Method(s): The methylation status of TIMP3 promoter in CRC cell lines, HT29, Vaco5 and Vaco411, was determined using methylation specific PCR. Conditioned media from these cell lines were assayed by ELISA to quantitate secreted TIMP3, soluble amphiregulin and soluble TNFR1. TIMP3 expression was also measured using quantitative RT-PCR. EGFR pathway activation was examined by measuring the levels of phospho-MEK by western blot. Result(s): The results show that in CRC cell lines, the methylation status of the TIMP3 promoter correlates with decreased TIMP3 expression, increased amphiregulin secretion and EGF pathway activation. The unmethylated TIMP3 promotor is associated with robust TIMP3 expression, decreased amphiregulin secretion and decreased EGF pathway activation. Furthermore, methylation of the TIMP3 promoter and decreased TIMP3 expression is associated with increased levels of soluble TNFR1. Conclusion(s): The methylation status of TIMP3 promoter may predict increased EGF pathway activation due to TIMP3 inactivation and subsequent increased EGFR ligand shedding, as well as inhibition of apoptotic signaling through TNFR1. These results suggest that TIMP3 methylation in colorectal cancers may result in increased EGFR pathway dependence through dual mechanisms and may therefore predict responsiveness to EGFR inhibitor therapy.