Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases

BACKGROUND: The family of proprotein convertases has been recently implicated in tumorigenesis and metastasis in animal models. However, these studies have not yet been completely corroborated in human tumors. METHODS: Using RT PCR, immunoblot and immunohistochemistry we assessed the presence and the processing patterns of the convertases PC1 and PC2 as well as the PC2 specific chaperone 7B2 in human liver metastases originating from colorectal cancer and compared them to unaffected and normal liver. Furthermore, we assessed the presence and processing profiles of PC1, PC2 and 7B2 in primary colon cancers. RESULTS: mRNA, protein expression, and protein cleavage profiles of proprotein convertases 1 and 2 are altered in liver colorectal metastasis, compared to unaffected and normal liver. Active PC1 protein is overexpressed in tumor, correlating with its mRNA profile. Moreover, the enhanced PC2 processing pattern in tumor correlates with the overexpression of its specific binding protein 7B2. These results were corroborated by immunohistochemistry. The specific and uniform convertase pattern observed in the metastases was present only in a fraction of primary colon cancers. CONCLUSION: The uniformly altered proprotein convertase profile in liver metastases is observed only in a fraction of primary colon cancers, suggesting possible selection processes involving PCs during metastasis as well as an active role of PCs in liver metastasis. In addition, the exclusive presence of 7B2 in metastatic tumors may represent a new target for early diagnosis, prognosis and/or treatment

Role of Pro-oncogenic Protein Disulfide Isomerase (PDI) Family Member Anterior Gradient 2 (AGR2) in the Control of Endoplasmic Reticulum Homeostasis

International audienc

Nck-dependent Activation of Extracellular Signal-regulated Kinase-1 and Regulation of Cell Survival during Endoplasmic Reticulum Stress

In response to stress, the endoplasmic reticulum (ER) signaling machinery triggers the inhibition of protein synthesis and up-regulation of genes whose products are involved in protein folding, cell cycle exit, and/or apoptosis. We demonstrate that the misfolding agents azetidine-2-carboxylic acid (Azc) and tunicamycin initiate signaling from the ER, resulting in the activation of Jun-N-terminal kinase, p44(MAPK)/extracellular signal-regulated kinase-1 (ERK-1), and p38(MAPK) through IRE1α-dependent mechanisms. To characterize the ER proximal signaling events involved, immuno-isolated ER membranes from rat fibroblasts treated with ER stress inducers were used to reconstitute the activation of the stress-activated protein kinase/mitogen-activate protein kinase (MAPK) pathways in vitro. This allowed us to demonstrate a role for the SH2/SH3 domain containing adaptor Nck in ERK-1 activation after Azc treatment. We also show both in vitro and in vivo that under basal conditions ER-associated Nck represses ERK-1 activation and that upon ER stress this pool of Nck dissociates from the ER membrane to allow ERK-1 activation. Moreover, under the same conditions, Nck-null cells elicit a stronger ERK-1 activation in response to Azc stress, thus, correlating with an enhanced survival phenotype. These data delineate a novel mechanism for the regulation of ER stress signaling to the MAPK pathway and demonstrate a critical role for Nck in ER stress and cell survival

Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases-5

<p><b>Copyright information:</b></p><p>Taken from "Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases"</p><p>BMC Cancer 2005;5():149-149.</p><p>Published online 17 Nov 2005</p><p>PMCID:PMC1310616.</p><p>Copyright © 2005 Tzimas et al; licensee BioMed Central Ltd.</p>identified. Light microscopy 7B2 immunohistochemistry of liver metastasis (T) and adjacent unaffected parenchyma (U), using 100 × magnification. Arrowheads indicate positively stained cells uniquely in the metastasis. Light microscopy 7B2 immunohistochemistry of liver metastasis (T) using 400 × magnification. Arrowheads indicate dense positively stained tumor cells

Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases-1

<p><b>Copyright information:</b></p><p>Taken from "Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases"</p><p>BMC Cancer 2005;5():149-149.</p><p>Published online 17 Nov 2005</p><p>PMCID:PMC1310616.</p><p>Copyright © 2005 Tzimas et al; licensee BioMed Central Ltd.</p>mples. Quantification was obtained by the ratio of the optical density of PC2 PCR amplification products over GAPDH. Standard error of mean (SEM) is indicated. Asterisk indicates a statistically significant difference (< 0.003). Schematic representation of PC2 structure. The antigenic region against which the antibody was raised is mentioned (YAb). Cleavage sites are indicated (arrows). PC2 cleavage profile in normal (N1-N3, top panel), unaffected (U1-U2) and tumor (T1-T2, bottom panel) samples. Normal liver samples (N1-N3) derived from three different organ donors. T1/U1 and T2/U2 came from two independent patients. PC1 immunoblotting was done on a total of 3 normal, 14 metastasis and 14 unaffected liver samples, and was repeated three times. Relative amounts of total PC2 (C, left panel) protein, in tumor (T), unaffected (U) and normal liver (N), expressed as a ratio over normal liver samples. Ratios of p75/p66 PC2 isoforms (C, right panel) in the same samples are calculated. SEM is indicated. Simple and double asterisks indicate statistically significant differences (P < 0.05)

Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases-6

<p><b>Copyright information:</b></p><p>Taken from "Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases"</p><p>BMC Cancer 2005;5():149-149.</p><p>Published online 17 Nov 2005</p><p>PMCID:PMC1310616.</p><p>Copyright © 2005 Tzimas et al; licensee BioMed Central Ltd.</p>fferent PC1 processing pattern between the two colon cancers. Experiments were repeated three times. Schematic representation of PC1 structure. PC2 immunoblots in primary colon cancer in unaffected colon and in an additional primary colon cancer. Note the absence of the 66 Kda band in the second primary colon cancer. Experiments were repeated three times. Schematic representation of PC2 structure. 7B2 immunoblots in primary colon cancer in unaffected colon and in an additional primary colon cancer, indicating presence of 7B2 protein only in one tumor. Experiments were repeated three times. Corresponding gels stained with Coomassie blue G250 (CS) are shown. Schematic representation of 7B2 structure

Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases-3

<p><b>Copyright information:</b></p><p>Taken from "Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases"</p><p>BMC Cancer 2005;5():149-149.</p><p>Published online 17 Nov 2005</p><p>PMCID:PMC1310616.</p><p>Copyright © 2005 Tzimas et al; licensee BioMed Central Ltd.</p>rcely positively stained hepatic cells. Light microscopy PC1 immunohistochemistry of liver metastasis (T) and adjacent unaffected parenchyma (U), using 20 × magnification. Arrowheads indicate positively stained tumor cells. Light microscopy PC1 immunohistochemistry of liver metastasis (T) using 400 × magnification. Arrowheads indicate positively stained tumor cells

Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases-4

<p><b>Copyright information:</b></p><p>Taken from "Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases"</p><p>BMC Cancer 2005;5():149-149.</p><p>Published online 17 Nov 2005</p><p>PMCID:PMC1310616.</p><p>Copyright © 2005 Tzimas et al; licensee BioMed Central Ltd.</p>itively stained hepatic cells. Light microscopy PC2 immunohistochemistry of liver metastasis (T) and adjacent unaffected parenchyma (U), using 25 × magnification. Arrowheads indicate positively stained cells mainly in the unaffected liver. Light microscopy PC2 immunohistochemistry of liver metastasis (T) using 200 × magnification. Arrowheads indicate positively stained tumor cells

Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases-2

<p><b>Copyright information:</b></p><p>Taken from "Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases"</p><p>BMC Cancer 2005;5():149-149.</p><p>Published online 17 Nov 2005</p><p>PMCID:PMC1310616.</p><p>Copyright © 2005 Tzimas et al; licensee BioMed Central Ltd.</p>mples. Quantification was obtained by the ratio of the optical density of 7B2 PCR amplification products over GAPDH. Standard error of mean (SEM) is indicated. Asterisk indicates a statistically significant difference (< 0.003). Schematic representation of 7B2 structure. The furin cleavage site is shown (arrow). The antigenic region against which the antibody was raised is indicated (YAb). 7B2 immunoblots in normal liver (N1-N3, top panel), in tumor (T1, T2, bottom panel) and unaffected (U1, U2, bottom panel) liver, indicating presence of 7B2 protein only in tumor. Corresponding gels stained with Coomassie blue G250 (CS) are shown. Experiments were repeated three times