Despite numerous biomarkers reported, few are useful for predicting metastasis. In our recent studies, 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. 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 120 stage I and stage II HCC patients is now in progress to further evaluate CPE-deltaN as a prognostic biomarker for predicting future metastasis. In another prospective clinical study on 42 patients with pheochromocytoma/paragangliomas, we were able to predict with high accuracy from the mRNA copy numbers of CPE-deltaN in the resected tumors, those patients who would develop future metastasis, even though they were diagnosed with benign tumors at the time of surgery. Additionally, in an ongoing prospective study of papillary thyroid cancer, CPE-deltaN was found to be an excellent biomarker for diagnosis of metastasis and identifying patients who have high or low risk of recurrence. Continued followup of these patients will substantiate our prediction. In a retrospective study on colorectal cancer, CPE-deltaN levels with a T/N >2 ratio, accurately diagnosed metastatic disease. We have also initiated a study on renal cell carcinoma (RCC). CPE-deltaN mRNA copy number was significantly elevated in metastatic RCC compared with benign samples and this was independent of RCC subtype, tumor size, age or gender. Furthermore, as a diagnostic biomarker, CPE-deltaN expression distinguished metastatic from benign with high specificity and sensitivity. Post surgery follow ups are now in progress to assess CPE-ΔN as a prognostic biomarker for predicting future metastasis. Thus, CPE-deltaN is a novel tumor inducer and a powerful prognostic marker for predicting future metastasis in different cancer types, superior to histopathological diagnosis. In endocine/neuroendocrine tumors, in addition to CPE-deltaN, wild-type CPE is highly expressed. We have studied the role of secreted WT-CPE in endocrine tumors using rat PC12 cells, a pheochromocytoma cell line, as a model. Anti-CPE antibodies added to the medium of PC12 cells to neutralize secreted CPE resulted in decreased survival compared to control cells under hypoxic and glucose deprivation conditions, suggesting that CPE promotes survival of these cells. To further study the role of WT-CPE in cancer cell survival, we used an epithelial derived hepatocellular carcinoma cell line, MHCC97H which does not have endogenous WT-CPE. Recombinant WT-CPE added to the medium of the HCC cells promoted survival of these cells compared to untreated cells under hypoxic and glucose starvation conditions. Hence WT-CPE, in addition to acting as a prohormone processing enzyme in neuroendocrine tumors, plays a role in promoting cell survival. Studies to understand the mechanism of action of extracellular WT-CPE indicate that it can activate the expression of the Bcl2 gene, an anti-apoptotic protein to enhance survival. In addition we found that WT-CPE has anti-migration and anti-invasion effects. WT-CPE has been shown to negatively regulate the Wnt pathway by forming a complex with the Wnt3a ligand and the Frizzled receptor. This complex then disrupts disheveled-induced signalosomes that are important for transducing the Wnt signal and reduces beta-catenin levels, a protein known to anchor to actin cytokeleton. Negative regulation of the Wnt pathway by WT-CPE may have a role in inhibiting tumor migration/invasion.
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