Abstract 5327: An N-terminal truncated carboxypeptidase E splice isoform induces metastasis by activating nedd9 and other metastasis inducing genes

Abstract

Abstract Cancer mortality is often from metastatic disease rather than the direct effect of the primary tumor. Identification of metastasis inducing genes may offer valuable mechanistic insight for guiding specific therapeutic strategies. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-ΔN) that drives metastasis. CPE-ΔN mRNA was elevated in human metastatic colon, breast and HCC cell lines. Suppression of CPE-ΔN expression in these cell lines by si-RNA significantly inhibited their growth and invasion. To confirm these observations in vivo, an orthotopic nude mouse model was established. The mice implanted with a tumor derived from HCC cells transfected with si- CPE-ΔN RNA in the liver did not show tumor growth or metastasis, compared to scrambled controls. In HCC cytosolic CPE-ΔN protein was translocated to the nucleus and upregulated the expression of neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9), through interaction with histone deacetylase (HDAC) 1/2. Inhibition of HDAC activity by the HDAC inhibitors suppressed expression of NEDD9, without effecting CPE-ΔN expression. The enhanced invasive phenotype of HCC cells stably transfected with CPE-ΔN was suppressed when Nedd9 was silenced by si-RNA. cDNA Microarray studies of HCC cells overexpressing CPE-ΔN showed elevated expression of 27 genes associated with metastasis such as Nedd9, claudin 2 (cldn2), carcinoembryonic antigen-related cell adhesion molecule 5 (ceacam5), matrix metallopeptidase 1 (mmp1), plasminogen activator (plat) and inositol 1,4,5-trisphosphate 3-kinase A (itpka), while 30 genes associated with tumor suppressor function, which included insulin-like growth factor binding protein 5 and 3 (igfbp5 and igfbp3) and h19 were down-regulated. These data were further confirmed by qRTPCR. Robust increase in the levels of protranscriptional acetylated histone H3 and H4 in these cells suggest epigenetic regulation of downstream genes by CPE-ΔN. In clinical studies, CPE-ΔN mRNA quantification in 99 patients with primary tumor (T) and normal surrounding tissue (N) established a T/N cut off value which predicted future intra-hepatic metastasis with high sensitivity and specificity, independent of cancer stage. Additionally, prospective study on 16 resected PHEO/PGLs having very high CPE-ΔN mRNA copy numbers accurately predicted future metastasis or recurrence in patients diagnosed with benign tumors at time of surgery, versus low copy numbers in post surgery, disease-free patients. Thus CPE-ΔN induces tumor metastasis and is a powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients, superior to histopathological diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5327. doi:1538-7445.AM2012-5327