AGR2 expression has previously been shown to be induced from the earliest precursor lesions of pancreatic cancer, PanINs, as well as in PDACs (9
). Here, we provide a comprehensive analysis of AGR2 expression in both pancreas and extrapancreatic tissues, showing that it is widely expressed in both sporadic and familial PanINs, PDACs, and metastatic lesions. In contrast, AGR2 is not widely expressed in normal organs, in which it is mostly confined to mucin-secreting cells (21
We show that in PDAC cells, AGR2 localizes to the ER, as observed in normal intestinal epithelial cells (12
), suggesting that AGR2 could exert a PDI activity on presecretory proteins also in transformed cells. Furthermore, we provide the first evidence that AGR2 also localizes to the external surface of AGR2-expressing pancreatic cancer cells; accumulation at the cell surface has previously been shown for several ER proteins (22
). Therefore, AGR2 may be used for the detection of circulating tumor cells in the peripheral blood of patients with pancreatic cancer and could also be a novel tumor cell surface antigen for the development of antibody-targeting strategies (24
). An increasing number of recent studies report that PDI proteins have important roles on the cell surface, as majority of surface proteins contain disulfide bonds (reviewed in refs. 23
) in which they can modulate the activity of membrane receptors (and thus activate and regulate signaling pathways; ref. 26
), adhesion molecules integrins (27
), or even proteases such as ADAM17 (28
). We and others show that AGR2 can also be found in cell culture supernatants (10
) and pancreatic juice (29
). It remains to be established whether AGR2 functions also at the cell surface or when secreted and whether it is immunogenic as shown for PDI proteins in renal cell carcinoma (30
Protein profiling of AGR2-expressing MiaPaCa2 cells identified upregulation of several ER chaperones (PDI, CALU, and RCN1), suggesting activation of the ER stress response. This correlates with a previous report on Agr2−/−
mice that showed that Agr2
is involved in the ER stress response as well as being itself induced by ER stress (31
). Stressful conditions such as hypoxia, nutrient deprivation, and pH changes encountered by tumor cells are known to induce ER stress (32
), characterized by the upregulation of ER chaperones. This enables cells to adapt to an unfavorable microenvironment and avoid ER stress–induced apoptosis. The main cellular PDI, PDI/P4HB, in which upregulation was seen here, is a well-established executor of the ER stress response and has been shown to protect melanoma cancer cells against ER stress–induced cell death (33
). CALU and RCN1 are EF-hand members of the CREC protein family (34
) which is associated with various Ca2
-dependent processes in the secretory pathway; in the ER, these proteins interact with the protein translocase (35
) that guides the transport of nascent presecretory proteins into the ER lumen. The regulation of these 2 ER chaperones by AGR2 could thus directly affect one of the very first steps in the protein secretion process (35
Interestingly, Denoyelle and colleagues (36
) showed that the induction of ER stress chaperones is an early event in the initiation of melanoma. Similarly, the universal AGR2 expression in all precursor lesions in both sporadic and familial settings suggests that activation of ER stress response could be one of the earliest events in PDAC development and could provide a survival advantage to premalignant cells. Although in the present study, we report on the consequences of AGR2 induction in the pancreas, the nature of the initial trigger that causes early AGR2 induction is not known and currently under investigation as it could potentially lead to a better understanding of pancreatic cancer initiation.
The expression of PDI proteins and ER chaperones has also been correlated with cancer invasion and metastasis in several tumor types (33
), and we here provide similar evidence for the role of AGR2 in PDAC.
AGR2 expression induced an increase in the levels of CTSB and CTSD, 2 disulfide-containing thiol proteases that have previously been reported to be upregulated in pancreatic cancer (16
) and are known to play a role in the dissemination of cancer cells (40
). Our in vitro
data indicated that AGR2-induced invasion was mediated through the action of these proteases rather than by increased cell motility, as no difference in migration and wound-healing assays was observed. The AGR2-induced increase in CTSB and CTSD levels could be the direct result of AGR2 PDI activity in the ER during the processing of pro-cathepsins, as previously reported for the production of MUC2 in enterocytes (12
), especially because we also observed an increase in the levels of cathepsin precursor forms. This is also in accordance with a number of other reports of PDI activity in protein folding being a limiting factor for protein synthesis and secretion (43
). The concept of a posttranslational regulation of cathepsins as observed previously (45
), and here reported to be mediated by AGR2, is additionally supported by the absence of change in cathepsins mRNA levels upon AGR2 upregulation. Identifying the formation of mixed disulfide bonds between AGR2 and CTSB and CTSD would confer a formal evidence of this mechanism in pancreatic cancer cells; however, the speed and the transient nature of this type of interaction potentiated by the presence of only 6 and 4 disulfide bonds in CTSB and CTSD, respectively (46
), rendered such experimental confirmation challenging. Our in vivo
studies using transparent zebrafish embryos, which provide an elegant short-term invasion assay and allows quantification of the DTCs with a single-cell resolution (47
), faithfully recapitulated in vitro
data and further substantiated that the role of AGR2 in increased invasion of pancreatic cancer cells is largely mediated by the 2 cathepsins.
Finally, we observed that AGR2 expression in PDAC cells is also involved in regulation of pro-CTSD secretion; overexpression of CTSD in cancer cells was reported previously to lead to the hypersecretion of its proteolytically inactive proform (48
). Furthermore, increased levels of pro-CTSD have been found in the plasma of patients with metastatic breast carcinoma (49
) and antibodies recognizing pro-CTSD in the serum of patients with ovarian cancer (50
). If a similar situation is established in PDAC, pro-CTSD might constitute a potential target for new detection strategies.
In summary, we provide new insights into the mechanisms of action of AGR2 and its role in dissemination of pancreatic cancer cells through regulation of CTSB and CTSD both in vitro and in vivo. In addition, we show that AGR2 can also be immunodetected at the surface of cancer cells, which could open new avenues for both the early detection and the development of novel immunotherapeutic strategies in pancreatic adenocarcinoma.