In this study, we assessed the presence and the cleavage/processing patterns of the two major convertases of the regulated secretory pathway, PC1 and PC2, as well as the PC2 chaperone 7B2. This study was performed in human liver metastases specimens from colorectal primaries and in unaffected liver samples from the same patients that underwent liver resection, as well as in normal livers. We also assessed these convertase profiles in primary colon cancers.
To our knowledge this is the first report describing the expression of PC1, PC2 and 7B2 in human liver tissues, and in human colon cancers. More importantly, we noted that (i) at the mRNA level, PC1 is overexpressed in metastatic tumor versus unaffected and normal liver, while PC2 expression is downregulated (Fig. 1A and 2A respectively); (ii) consistently, at the protein level, PC1 (p84+p66) is overexpressed (Fig. 1D), while PC2 (p75+p66) is downregulated in metastatic tumor (Fig. 2D); (iii) both active PC1 and PC2 are predominant in metastatic tumor (Fig. 1D and 2D respectively); (iv) consistently with the enhanced PC2 zymogen processing pattern, in tumor, 7B2 is overexpressed (Fig. 3A and 3C); (v) the above results are corroborated by immunohistochemistry (Fig. 4, 5, 6).
We also found that the specific PC2 and 7B2 profile observed in metastatic cancers was observed only in a fraction of primary colon cancers. These data support a specific negative feedback mechanism regulating PC2 mRNA expression in liver metastases, when PC2 proteolytic activity is overwhelming. Therefore in metastatic tumors, abundant active PC2 may lead to a downregulation of its mRNA, and vice versa in unaffected and normal liver. Furthermore, the homogeneous liver metastasis PC2 protein profile supports the hypothesis of a tightly regulated active PC2 production in the primary as well as in metastatic tumors, with 7B2 playing a leading role in this mechanism. Interestingly, PC1, PC2 and 7B2 are considered as markers of endocrine and neuroendocrine phenotypes [5, 8, 12]. The fact that they are also detected in human anal canal  from which also cancers and eventually metastases can develop suggests that a neuroendocrine differentiation program could take place during colon carcinogenesis and liver metastasis. Recently, a specific RE1-lk silencer element in the promoter of PC2 was identified  and binding of transcription-silencing factors to this element may contribute to repression of the PC2 gene in non-neuroendocrine cells.
Our results are in agreement with recent experimental animal evidence highlighting the potential implication of convertases in tumorigenesis and metastasis. Indeed, Khatib et al. have shown that convertase inhibition in the HT-29 colon cancer cell line is followed by decreased invasiveness and tumorigenicity . In addition it has been shown that furin, another member of the convertase family, is implicated in tumor progression in human head and neck malignancies . Convertase overexpression can also alter the growth behavior and the drug responsiveness in a human breast cancer cell line model . In the past, 7B2 has been implicated with several types of neuroendocrine tumors, such as neuroendocrine bronchial tumors, nonfunctioning pancreatic islet tumors and ACTH-secreting pituitary tumors [17, 18], mainly participating at the processing of tumor-secreted active peptides. However its role in tumorigenesis and metastasis, especially in colorectal cancer, remains largely unknown.
Other groups have also recently showed the implication of several members of the convertase family in the carcinogenesis process. Specifically Siegfried et al have clearly shown that members of the convertase family process VEGF-C, a known tumorigenic growth factor, in animal models . The same group has demonstrated that convertases are involved in the processing of pro-platelet derived growth factor A, a hallmark of carcinogenesis , thus concluding that convertase inhibitors might be used eventually in the treatment of neoplasia.
Our data support the accumulation of active PC1 and PC2 in metastasis, and are in agreement with previous reports [3, 16]. The main question although remains whether alterations of PC1, PC2 and 7B2 expression profiles are the cause or consequence of the metastatic phenotype. PC1 and PC2 process pro-neurotensin to its active form, which has been involved in colonic tumorigenesis [21, 22], or pro-pancreatic peptide, proGHRH, proglucagon, prosomatostatin, and pro-insulin to insulin [23, 24], which are known trophic factors for the gut and possibly involved in tumorigenesis as well. Indeed, recently it was shown that PC1 null mice are dwarfed, thus implicating PC1 in the processing of GHRH and subsequent growth . Therefore change in PC1 and PC2 expression and activation may alter the profiles of secretory proteins, which in turn could increase cell growth potential. These changes could lead to selection of primary colon cancer cells destined to metastasize to the liver or even render the rest of the liver susceptible to future/further metastasis. Indeed, the fact that the PC2 profile observed in unaffected liver is partially different from the one observed in normal liver, supports the hypothesis that the tumor may induce modifications in the rest of the liver. Whether PC1, PC2 and 7B2 are directly implicated in such a model is under investigation, and it would be interesting also to evaluate the profile of these convertases in different also metastatic sites such as in the lung or in the brain.
In conclusion, the present study shows that PC1 and PC2 convertase expression and cleavage are altered in CRC liver metastases. 7B2, whose overexpression in tumor is thought to play a key role in the above processes, could represent a potential diagnostic, prognostic or even therapeutic target.
Furthermore, the metastasis/tumor associated convertase profile is observed only in a fraction of primary colon cancers thus suggesting a potential selection process for tumors that eventually will develop metastasis and may be associated with worse clinical outcome. These observations require prospective validation that is underway.