Despite numerous biomarkers reported, few are useful for predicting metastasis. We have uncovered a novel splice isoform of the prohormone processing enzyme, carboxypeptidase E (CPE-deltaN) that is elevated in metastatic hepatocellular, colon, breast, head and neck carcinoma. CPE-deltaN lacks the N-terminus that is normally found in wild-type CPE. Wild-type CPE is localized in secretory granules, however, CPE-deltaN is translocated from the cytoplasm to the nucleus of metastatic cancer cells. Overexpression of CPE-deltaN in hepatocellular carcinoma (HCC) cells promoted their proliferation and migration. SiRNA knockdown of CPE-deltaN expression in highly metastatic HCC cells inhibited their growth and metastasis in nude mice. CPE-deltaN promoted migration by up-regulating expression of the metastasis gene, Nedd9, through interaction with histone deacetylase (HDAC) 1/2, whereas inhibition of HDAC activity by the HDAC inhibitors suppressed expression of NEDD9. The enhanced invasive phenotype of HCC cells stably transfected with CPE-deltaN was suppressed when Nedd9 was silenced by siRNA. We further investigated what other genes might be modulated by CPE-deltaN. Microarray studies of HCC cells overexpressing CPE-deltaN showed elevated expression of 27 genes associated with metastasis including Nedd9, claudin 2 (cldn2), 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, such as insulin-like growth factor binding protein 5 and 3 (igfbp5 and igfbp3) and h19 were down-regulated. In a recent study, we have shown that CPE-deltaN can activate the canonical Wnt pathway resulting in increased levels of beta-catenin which functions with T-cell factor/lymphoid enhancer factor in the nucleus to activate expression of Wnt target genes. It is well known that such a mechanism could lead to colorectal cancer progression. We have also examined the extracellular role and mechanism of action of CPE in tumor cell growth and survival from several cancer types using purified recombinant CPE. We showed that rat pheochromocytoma cells, a neuroendocrine tumor cell line (PC12) secretes CPE and addition of an anti-CPE neutralizing antibody in the cell medium resulted in increased cytotoxic effects and poor survival of the cells under metabolic stress (nutrient starvation and hypoxia). This loss of function experiment demonstrates that CPE is involved in maintaining the durability and resilience of neuroendocrine tumors under this type of stress. In gain of function experiments, we found that HCC cells, that do not synthesize much CPE, and showed significantly less cytotoxicity under these metabolic stress conditions when purified recombinant CPE protein was added to the culture medium. This effect was also observed when CPE was treated with 5microM GEMSA, a specific and potent inhibitor of CPE, indicating that the extracellular role of CPE in imparting resistance to the cells during metabolic stress is independent of its enzymatic activity. We found that treatment of HCC cells under metabolic stress, with CPE, resulted in increased phosphorylation of ERK1/2 and an increase in the expression of the survival gene BCL-2, at the mRNA and protein levels. Thus CPE is a tumor pro-survival factor during metabolic stress, acting through ERK-signaling. In addition, CPE treatment caused an increase of phospho-GSK3beta; (Ser9) and active-beta;-catenin, suggesting the involvement of the canonical Wnt signaling pathway. Several other genes (TNF, NF-kappa,beta, I-kappa,beta,alpha, and IL-8), which could support tumor cell survival were also up-regulated in the CPE-treated HCC cells under metabolic stress. We also demonstrated the ability of extracellular CPE to inhibit migration and invasion of a very aggressive fibrosarcoma cell line, HT1080, suggesting that CPE has anti-metastatic effects in these cells. The mechanism underlying the inhibition of migration or invasion by CPE is less clear. Since the Wnt pathway components can mediate cancer cell invasion, one can speculate that the negative regulation of the Wnt pathway by CPE that we reported previously could be responsible for the inhibition of migration and invasion observed with CPE treatment. Our studies also indicated that CPE can drive tumor cell survival through ERK-BCL-2 signaling, as well as activate the wnt pathway during metabolic stress. Thus CPE can have different effects in different cancer cell types. Hence, the level of expression of CPE, the tumor environment and contributions from other pathways, all dictate the final phenotype of the tumor (Murthy et al, Cancer Lett. 2013). In retrospective clinical studies of 180 patients with HCC, CPE-deltaN mRNA quantification in primary HCC tumor (T) versus surrounding normal tissue (N) established a T/N cut off level, above which predicted future metastasis within 2 years with high sensitivity and specificity and independent of cancer stage. A prospective study, on 130 stage I and stage II lung adenocarcinoma cancer patients further suggests that CPE-deltaN mRNA is a good prognostic biomarker for predicting future metastasis with high sensitivity for stage I and II patients. Additionally, in an ongoing prospective study of papillary thyroid cancer, CPE/CPE-deltaN mRNA was found to be an excellent biomarker for diagnosis of identifying patients who have high risk of recurrence. Continued followup of these patients will substantiate our prediction. In a retrospective study on colorectal cancer, CPE-deltaN mRNA levels with a T/N >2 ratio, accurately diagnosed metastatic disease. Thus, CPE/CPE-deltaN is a powerful prognostic marker for predicting future metastasis in different cancer types, superior to histopathological diagnosis. Recently, we have developed an assay for detecting CPE/CPE-deltaN in serum. We can assay quantitatively for CPE/CPE-deltaN mRNA in exosomes prepared from serum. Preliminary results indicate a highly significant difference in serum levels of CPE/CPE-deltaN mRNA between normal and cancer patients. This will provide a non-invasive method for screening for cancer in high risk patients.
