Site-dependent E-cadherin cleavage and nuclear translocation in a metastatic colorectal cancer model

Abstract

Metastases are frequently found during colorectal cancer diagnoses and are the main determinants of clinical outcome. The lack of reliable models of metastases has precluded their mechanistic understanding and our capacity to improve outcome. We studied the effect of E-cadherin and Snail1 expression on metastagenesis in a colorectal cancer model. We microinjected SW480-ADH human colorectal cancer cells, transfected with an empty vector (Mock) or overexpressing Snail1 (Snail1(OE)) or E-cadherin (E-cadherin(OE)), in the ceca of nude mice (eight per group) and analyzed tumor growth, dissemination, and Snail1, E-cadherin, β-catenin, and Presenilin1 (PS1) expression in local tumors and/or metastatic foci. Snail1(OE) cells disseminated only to lymph nodes, whereas Mock or E-cadherin(OE) cells spread to lymph nodes and peritoneums. Peritoneal tumor foci developed by E-cadherin(OE) cells presented an increase in E-cadherin proteolysis and nuclear translocation, and enhanced expression of proteolytically active PS1, which was linked to increased tumor growth and shortened mouse survival. Interestingly, local and lymph node tumors in mice bearing E-cadherin(OE) cells overexpressed E-cadherin, but they did not show E-cadherin proteolysis or nuclear translocation. Remarkably, E-cadherin nuclear translocation and enhanced expression of active PS1 were found in a patient with colorectal signet-ring cell carcinoma. In conclusion, we have established a colorectal cancer metastasis model in which E-cadherin proteolyis and nuclear translocation associates with aggressive foci growth only in the peritoneal microenvironment.

Figure 1

Characterization of cultured human SW480-ADH colorectal cancer cells stably expressing an empty vector (Mock cells) or overexpressing Snail1 (Snail1^OE cells) or E-cadherin (E-cadherin^OE). These cells were previously generated (22 and 25). A–C: Phase-contrast microscopy images of Mock (A), Snail1^OE (B), and E-cadherin^OE (C) SW480-ADH cells. Scale bar = 50 μm. Snail1^OE cells displayed a large and stellate phenotype with few cell-cell contacts. E-cadherin^OE cells displayed an epitheloid morphology with higher cell density and cell–cell contacts. Mock cells displayed a mixed phenotype with an intermediate cell density and a few suspended cells. D and E: The expression of the mesenchymal markers LEF1 and Fibronectin, as measured by Western blotting (D) or qRT-PCR (E), was higher in Snail1^OE than in Mock or E-cadherin^OE cells. E-cadherin^OE cells showed a complete shut-down of LEF1 expression. F: β-catenin/TCF transcriptional activity was extremely reduced in E-cadherin^OE cells compared with Mock or Snail1^OE cells. G: β-catenin was nuclear (DAPI staining colocalization) in Mock and Snail1^OE cells, whereas it was membranous and cytosolic in E-cadherin^OE cells. Scale bar = 50 μm. H: E-cadherin was mostly membranous in E-cadherin^OE cells. Scale bar = 50 μm.

Figure 2

Histopathology in local colonic tumors and lymph node foci derived from Mock, Snail1^OE, and E-cadherin^OE cells. Local tumors were poorly differentiated in the Mock group (A), undifferentiated in the Snail1^OE group (B), and well-differentiated, containing glands and mucous deposits (black arrow) in the E-cadherin^OE group (C). The mitotic rate (white arrows) was similar in all three groups. All lymph node tumor foci (white asterisk) infiltrated the lymph nodes, pushing aside normal lymphocytes (white circle). The lymph node tumor foci were poorly differentiated in the Mock group (D), undifferentiated in the Snail1^OE group (E), and showed differentiation features in E-cadherin^OE group (F).

Figure 3

Peritoneal carcinomatotic foci development and survival in mice implanted with Mock or E-cadherin^OE cells. Tumor foci developed in the peritoneum (black arrow) after cecal implantation of Mock (A) or E-cadherin^OE (B) cells, but not after implantation of Snail1^OE cells. Tumor foci derived from E-cadherin^OE cells were large (B) and well-differentiated and they had a high mitotic rate (D, black arrows). Tumor foci in the Mock group were small (A) and poorly differentiated, and they had a lower mitotic rate (C, black arrows). E: Cumulative survival was significantly higher in mice bearing tumors derived from Mock or from Snail1^OE cells than in those bearing tumors derived from E-cadherin^OE cells.

Figure 4

Snail1 expression in local colonic tumors and lymph node foci generated from Mock, Snail1^OE, or E-cadherin^OE cells. Snail1 immunostaining in both local tumor (A–C) and lymph node foci (D–F) were weak and nuclear in the Mock group (A and D), very strong and mostly nuclear in the Snail1^OE group (B and E), and undetectable in the E-cadherin ^OE group (C and F). Black asterisk indicates tumor tissue; black circle indicates normal tissue.

Figure 5

E-cadherin and β-catenin expression in local colonic tumors derived from Mock, Snail1^OE, and E-cadherin^OE cells. E-cadherin immunostaining was weak and membranous in Mock group tumors (A), absent in Snail1^OE group tumors (B), and intense, cytosolic, and membranous in tumors derived from E-cadherin^OE cells (C). β-catenin immunostaining was moderately intense and nuclear in Mock group tumors (D), very intense and mostly nuclear in Snail1^OE group tumors (E), and weak and cytosolic in E-cadherin^OE group tumors (F). Hematoxylin costaining.

Figure 6

E-cadherin and β-catenin expression in lymph node foci derived from Mock, Snail1^OE, or E-cadherin^OE cells. E-cadherin immunostaining was weak and membranous in foci of the Mock group (A), undetectable in the Snail1^OE group (B), and cytosolic and membranous in the E-cadherin^OE group (C). β-catenin immunostaining was very strong and mostly nuclear in foci of the Mock (D) or Snail1^OE (E) groups, and weak, cytosolic, and membranous in foci of the E-cadherin^OE group (F). Hematoxylin costaining.

Figure 7

Snail1, E-cadherin, and β-catenin expression in peritoneal carcinomatotic foci of Mock and E-cadherin^OE mice. Snail1 immunostaining was weak and cytosolic in Mock (A) and undetectable in E-cadherin^OE (B) foci. E-cadherin immunostaining in peritoneal foci was weak and membranous in the Mock group (C) but very intense and mostly nuclear in the E-cadherin^OE group (D). β-catenin immunostaining in carcinomatotic foci was very strong and mostly nuclear in the Mock group (E) but weak and cytosolic in the E-cadherin^OE group (F). Hematoxylin costaining. White asterisks indicate tumor tissue.

Figure 8

Increased levels of proteolyzed E-cadherin and proteolytically active Presenilin1 (PS1) in peritoneal carcinomatotic foci developed from E-cadherin^OE cells. Peritoneal foci in the E-cadherin^OE group overexpressed E-cadherin and showed a significantly (P < 0.005) higher level of proteolyzed E-cadherin than peritoneal foci in the Mock group (A, left and B), than E-cadherin^OE local colonic tumors (A, center and C) or E-cadherin^OE cultured cells (A, right and C). Neither Mock nor Snail1 cultured cells showed E-cadherin proteolysis (A, right). PS1 was barely detectable in Mock, Snail1^OE, and E-cadherin^OE cells cultured in vitro (D). Moreover, peritoneal foci generated from E-cadherin^OE cells showed a significantly (**P < 0.01) higher ratio of active PS1 than foci derived from Mock cells (D and E). Ratio of proteolyzed E-cadherin = CF/(FL + CF); CF = 38-kDa cytosolic E-cadherin fragment; FL = 120 kDa full-length E-cadherin. Ratio of proteolytically active PS1 = pPS1/(npPS1 + pPS1); npPS1 = non-processed inactive 50 kDa PS1; pPS1 = processed and proteolytically active 29 kDa PS1; Control C1 = CHO k1 p70 cells; C2 = PC12 cells; there is statistically significant differences between groups **P < 0.01.

Figure 9

Nuclear E-cadherin and Presenilin1 (PS1) expression in peritoneal foci of colorectal signet-ring cell carcinoma. Two patients were analyzed. In the primary tumor and the carcinomatotic foci of one patient, there were moderate levels of E-cadherin in the membrane (A), intense and mostly nuclear β-catenin expression levels (B), and low PS1 RNA levels (E, qRT-PCR). In the second patient, we observed the same pattern of E-cadherin and β-catenin expression in the primary tumor, but in contrast, the carcinomatotic foci showed intense nuclear E-cadherin immunostaining (C), a complete absence of β-catenin expression (D), and a 13-fold increase in PS1 RNA expression (E).