Abstract 5162: Carboxypeptidase E splice isoform is a novel tumor inducer and biomarker for predicting future metastasis in human cancers

Abstract

Abstract Metastasis is a major cause of mortality in cancer patients, but mechanisms governing the metastatic process remain elusive. Furthermore, few accurate biomarkers exist for predicting future metastasis to guide therapy, especially for hepatocellular carcinoma (HCC) and pheochromocytomas /paragangliomas (PHEO/PGL), rare neuroendocrine tumors. We report that an alternatively spliced isoform of the prohormone processing enzyme, carboxypeptidase E (CPE-DELTA-N) drives metastasis. CPE-DELTA-N mRNA is 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 and invasion. Over-expression of CPE-DELTA-N in HCC cells promoted their proliferation and migration. cDNA Microarray studies showed that NEDD9 metastasis gene was up-regulated in these cells, which also exhibited elevated levels of NEDD9 protein and promoted their proliferation and invasion. This enhanced invasion was suppressed when NEDD9 was down regulated by si-RNA in stably CPE-DELTA-N expressing HCC cells. Bioinformatic studies identified a domain homologous to histone deacetylase (HDAC) interacting proteins in CPE-DELTA-N and co-immunoprecipitation studies indicated CPE-DELTA-N interacts with HDAC1/2. Inhibition of HDAC activity by the HDAC inhibitors suppressed expression of NEDD9 in HCC cells stably expressing CPE-DELTA-N, while there was no effect on CPE-DELTA-N expression. Immunofluorescence microscopy of metastatic MHCCLM3 cells revealed cytosolic CPE-DELTA-N were translocated to the nucleus. In vivo animal studies, nude mice were subcutaneously injected with MHCCLM3 cells transduced with either si- CPE-DELTA-N or si-scr controls. After thirty days, control mice had liver tumors 16 fold bigger in size than mice injected with si- CPE-DELTA-N cells. In a metastatic orthotopic nude mouse model, thirty-five days post-implantation into the liver of nude mice with the si-scr, MHCCLM3 derived tumors were 14 fold larger in size and developed intrahepatic metastasis and extrahepatic metastasis to lung compared to those from control cells. 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. 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: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5162. doi:10.1158/1538-7445.AM2011-5162