Abstract 3877: An N-terminal truncated carboxypeptidase E splice isoform induces metastasis by activating nedd9, ceacam5, cbr1 and other metastasis inducing genes.
Authors: Murthy Saravana R. , Lee Terence K. , Cawley Niamh X. , Hewitt Stephen M. , Pacak Karel , Loh Peng Y. 
Published: August 2014

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-DELTA-N) that drives metastasis. CPE-DELTA-N mRNA was elevated in human metastatic colon, breast and HCC cell lines. Suppression of CPE-DELTA-N expression in these cell lines by si-RNA significantly inhibited their growth migration 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-DELTA-N RNA in the liver did not show tumor growth or metastasis, compared to scrambled controls. In HCC cytosolic CPE-DELTA-N 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-DELTA-N expression. The enhanced invasive phenotype of HCC cells stably transfected with CPE-DELTA-N was suppressed when Nedd9 was silenced by si-RNA. cDNA Microarray studies of HCC cells overexpressing CPE-DELTA-N showed elevated expression of 27 genes associated with metastasis such as Nedd9, claudin 2 (cldn2), carcinoembryonic antigen-related cell adhesion molecule 5 (ceacam5) and carbonyl reductase 1(cbr1), 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. Suppression of CPE-DELTA-N expression in HT1080 cell line by si-RNA significantly down regulated ceacam5 and cbr1. In clinical studies, CPE-DELTA-N 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-DELTA-N 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. Additionally, CPE-DELTA-N is a diagnostic biomarker for metastasis in papillary thyroid carcinoma and colorectal cancer. Thus CPE-DELTA-N 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: Saravana R. Murthy, Terence K. Lee, Niamh X. Cawley, Stephen M. Hewitt, Karel Pacak, Peng Y. Loh. An N-terminal truncated carboxypeptidase E splice isoform induces metastasis by activating nedd9, ceacam5, cbr1 and other metastasis inducing genes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3877. doi:10.1158/1538-7445.AM2013-3877