Poster Sessions

CANCER-34

Saravana Murthy

S.R.K Murthy, T.K. Lee, N.X. Cawley, S.M. Hewitt, Y.P. Loh

A Splice Isoform of Carboxypeptidase E as a Biomarker for Predicting Metastasis in Human Cancers

Metastasis is a major cause of mortality in patients with cancer. Identifying molecules that promote tumor metastasis will elucidate complex mechanisms of metastasis. Such molecules can serve as biomarkers, improving prognosis and treatment for metastasis. To date only few biomarkers are useful for predicting metastasis especially for prevalent cancers such as human hepatocellular carcinoma (HCC). By bioinformatics analysis, we discovered a novel splice isoform of the prohormone processing enzyme, carboxypeptidase E (CPE-DELTA-N) in non-redundant nucleotide sequence EST database. CPE-DELTA-N lacks the N-terminus present in secretory granule wild-type CPE and immunofluorescence microscopy of metastatic MHCCLM3 cells revealed CPE-DELTA-N primarily in the nucleus. It is elevated in epithelial-derived metastatic colon, breast and prostate cancer cells. 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 by up-regulating NEDD9 metastasis gene expression. In vivo animal studies were carried out using two models. In one, nude mice were subcutaneously injected with MHCCLM3 cells transduced with either si- CPE-DELTA-N or si-scr. After thirty days, control mice bearing the si-scr MHCCLM3 cells 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 RNA quantification in primary HCC and colon tumors from patients established a level, above which predicted metastasis with high prognostic significance (p< 0.0001). The Kaplan-Meier plot of disease-free survival of 99 HCC patients and 68 colon cancer patients showed shorter survival times when CPE-DELTA-N mRNA were >2-fold (high) in the primary tumor compared to patients with less than 2-fold (low). Immunohistochemistry of CPE-DELTA-N in HCC tumors from 37 patients revealed immunostaining in the nuclei of tumor cells in patients who subsequently developed recurrence, but absent in controls. Thus, CPE-DELTA-N is a new mediator of tumor metastasis, a powerful prognostic marker and potential molecular target for therapy.