Northern blot analysis revealed two carboxypeptidase E (CPE) mRNAs (2.4 and 1.7 kb in size) present in human metastatic hepatocellular carcinoma (HCC), ovarian, glioma, lung and pancreatic cell lines. We have cloned both these mRNAs from HCC97H cells. The 2.4 kb mRNA represents CPE-WT and the 1.7kb is a novel mRNA encoding a 40kD N-terminal truncated splice isoform of CPE, we named 40kD CPE-deltaN. Western blot analysis of these cancer cell lines shows expression of the 40kD CPE-deltaN which lacks the N-terminal secretory pathway sorting signal, and is translocated from the cytoplasm to the nucleus. However wild-type CPE (WT-CPE) mRNA is poorly translated into protein in these cancer lines except in glioma cells, and it is mainly secreted into the medium. In HCC97H and Panc-1 pancreatic cancer cells, siCPE-RNA knockdown of all forms of endogenous CPE inhibited cell proliferation, migration and invasion. Ectopic expression of 40kD N-terminal truncated CPE-deltaN in HCC and Panc-1 cells promoted proliferation, colony formation and invasion, whereas overexpression of WT-CPE promoted proliferation but not invasion. We have examined the mechanism underlying the promotion of invasion in HCC and Panc-1 cells by 40kD CPE-deltaN and found that several metastasis-related genes such as CXCL12, CXCR2, CXCR4 and MMP3 were up-regulated when CPE-deltaN was overexpressed in these cells. These findings suggest that the 40kD CPE- deltaN plays an important role in cancer cell growth and metastasis through up-regulating expression of metastasis-related genes (10.18632/genesandcancer,193. (2019)). In collaboration with Dr. Arbersfeld (Tel Aviv University), we have shown that 40kD CPE-deltaN can activate the canonical Wnt pathway in HEK293 cells, resulting in increased beta-catenin expression. Beta-catenin functions with T-cell factor/lymphoid enhancer factor in the nucleus to activate expression of Wnt target genes, some of which are known to promote cancer cell proliferation. It is well known that such a mechanism involving the wnt pathway could lead to colorectal cancer progression. We have also examined the extracellular role and mechanism of action of CPE-WT 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, 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 pro-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 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. Such an effect has been reported for CPE-WT secreted from glioma cell lines. However, this anti-metastatic effect was not observed in HCC cells which synthesize and secrete only low levels of CPE-WT. The mechanism underlying the inhibition of migration or invasion by CPE is unclear. 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-WT can drive tumor cell survival through ERK-BCL-2 signaling, as well as activate the wnt pathway during metabolic stress. However, it does not appear to be involved in invasion, and in glioma and HT1080 cells even have anti-invasion effects, unlike CPE-deltaN. Thus CPE-WT can have different effects in different cancer cell types. Hence, the level of expression of CPE and CPE-deltaN, the tumor environment and contributions from other pathways, all dictate the final phenotype of the tumor. Exosomes represent a class of nano-sized extracellular vesicles, which are widely associated with cancer progression and metastasis. They carry biomolecules (proteins, DNA, mRNA and miRNA) unique to their cell of origin and deliver them to recipient target cells, thereby mediating cell-cell communication. Recently, we have developed an assay that can quantitatively measure CPE/CPE-deltaN mRNA copy numbers in exosomes prepared from cell culture media. Our results on HCC, pancreatic, ovarian and cervical cancer cell lines show significantly elevated levels of CPE/CPEdeltaN mRNA in high versus low metastatic cells of these different cancer types, although the size and exosome numbers were no different for high and low metastatic cells. Content analysis of exosomes derived from HCC high-metastatic (HCC97H) cells revealed presence of full length CPE-WT protein by Western Blot and CPE-WT mRNA using semi-quantitative PCR. We showed that exosomes released from HCC97H cells were able to enhance invasion and proliferation of HCC cells with poor metastatic ability (HCC97L). However, when CPE expression was suppressed in the HCC97H cells before exosome isolation, the exosomes had no effect on proliferation and invasion. These data show the ability of exosomes to confer metastasis in cancer cells and the role of exosomal CPE in driving the process. Since our previous studies have shown that down-regulation of CPE expression by CPE shRNA can reverse tumor growth and metastasis in a HCC mouse model, our future studies will focus on using exosomes as a vehicle for delivering CPE shRNA into cancers cells to inhibit growth and metastasis. This could potentially be developed as a new therapeutic approach to treating cancer.

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