Smad7 induces hepatic metastasis in colorectal cancer

Abstract

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-beta type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-beta/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.

Figure 1

Stable expression of Smad7 induces liver metastasis of colon adenocarcinoma (FET) cells after splenic injection in athymic nude mice. (A) Cells from each pool of parental FET, vector control and Smad7 overexpressing cells were injected into spleen of athymic nude mice. Mice were sacrificed 33 days after injection, liver tissues were separated and pictures were taken. (B) Represents total number of mice in each group used for splenic injection and total number of mice showing liver metastases. The average body weight and the average weight of liver tissues from each group with respective standard deviation were shown. (C) The graph shows the average of the ratio of liver weight and total body weight of each mouse in each group after metastases to the liver. (D) The PCR analyses were performed for hygromycin gene using genomic DNA from parental FET cells, vector control clone and three stable Smad7 clones as well as from the normal liver and liver metastases generated using these cell lines (top panel). Expression of Smad7 in liver metastasis was verified by western blotting using lysates prepared from normal livers of mice injected with FET cells and vector control clone as well as liver metastases generated by stable Smad7 clones (bottom panel).

Figure 2

Changes in protein expression in liver metastases. (A) Lysates were prepared from normal livers of mice injected with FET cells and vector control clone as well as liver metastases generated by stable Smad7 clones. Western blot analyses were performed using antibodies against TβRII, phospho-Smad2, Smad2, Smad3 and Smad4. β-actin was used as loading control. (B) Non-Smad pathway proteins were analysed by western blotting using antibodies against phospho-AKT, AKT, phospho-ERK and ERK. β-actin was used as loading control. (C) Junctional proteins such as E-cadherin, β-catenin, ZO-1, Claudin-1, Claudin-4, Claudin-7 and nm23, as well as uPA and p53 in normal and metastatic livers, were analysed by western blotting. β-actin was used as loading control. (D) The expression of cell cycle regulatory proteins such as p21^Cip1, p27^Kip1, Cdk2, Cdk4, Cyclin D1, phospho-Rb and Rb were analysed by western blotting. β-actin was used as loading control.

Figure 3

Correlation between higher levels of protein expression and aggressiveness of liver metastasis. (A) Lysates from livers with different degree of metastasis and unaffected normal liver tissues were tested for western blot analysis using antibodies against p53, E-cadherin, nm23, Claudin-4 and Claudin-7. β-actin was used as loading control. Aggressiveness in liver metastasis was assessed by the number of tumour nodules, volume of the liver occupied by tumour and total weight of the liver 33 days after splenic injection. (B) Intensity of protein bands for each tumour from mice in each group (n=5) with different degree of metastasis were calculated using TotalLab TL 100 software. The relative average band intensity for each protein from tumours with different degree of aggressiveness in liver metastasis was plotted. Data were presented as the mean±s.e. of five different tumours per group. The P-values were calculated by two-way ANOVA method using GraphPad Prism 4 software. ^*P<0.0001; ^**P<0.005; ^***P<0.001 by comparison with corresponding control values were shown. (C) The graph shows the percentage of total number of mice, with or without liver metastases, inoculated with vector control cells or Smad7-expressing cells. Percentage in the parenthesis indicates the aggressiveness in liver metastasis assessed as above.

Figure 4

Localisation of junctional proteins and phospho-Smad2 in Smad7-induced liver metastases. Tissues from normal livers of parental and vector control cells injected mice and liver metastases from Smad7-expressing clones injected mice were collected 33 days after splenic injection. (A) Shows staining with H&E, proliferation marker Ki67 and Tunnel. (B) Shows immunohistochemical staining for phospho-Smad2, E-cadherin, Claudin-1 and Claudin-4. Pictures were taken at original magnification of 630 × .

Figure 5

A model showing the role of Smad7 signalling in colon cancer metastasis. The TGF-β activates TβR heterotetrameric complex, leading to activation of the classical Smad2, Smad3 and Smad4 signalling cascade that induces cyclin-dependent kinase inhibitor p21^Cip1 and suppresses pro-oncogenic c-Myc expression. The p21^Cip1 expression leads to inhibition of cyclin-dependent kinases (CDKs) and cyclins that results in hypophosphorylation of Rb, repression of E2F transcriptional activity and inhibition of cell cycle progression. Smad7 negatively regulates TGF-β/Smad signalling pathways to induce cell proliferation by suppressing p21^Cip1 and by induction of c-Myc. Smad7 induces cell survival through the activation of AKT and inhibits apoptosis through the induction of TRX (thioredoxin-1) and ASK1 (apoptosis signal-regulating kinase-1). Smad7 also cooperates with activated Ras and induces tumorigenicity. All of these deregulations of cell behaviour may finally contribute to metastasis. Smad7 expression is induced by TGF-β, EGF, TNF-α and IFN-γ.