Epithelial-mesenchymal transition induced by growth suppressor p12CDK2-AP1 promotes tumor cell local invasion but suppresses distant colony growth

Abstract

Epithelial-mesenchymal transition (EMT) has been considered essential for metastasis, a multistep process including local invasion, intravasation, extravasation, and proliferation at distant sites. However, controversy remains as to whether EMT truly happens and how important it is to metastasis. We studied the involvement of EMT in individual steps of metastasis and found that p12(CDK2-AP1), a down-stream effector of transforming growth factor beta, induced EMT of hamster cheek pouch carcinoma-1 cells by promoting the expression of Twist2. EMT cells have an increased invasive but decreased metastatic phenotype. When s.c. inoculated, both EMT and non-EMT cells established primary tumors, but only EMT cells invaded into the adjacent tissues and blood vessels; however, neither cells formed lung metastases. When i.v. inoculated, only non-EMT cells established lung metastases. Moreover, s.c. inoculation of a mixture of the two cell types resulted in intravasation of both cell types and formation of lung metastasis from non-EMT cells. Our results allowed us to propose a novel model for the role of EMT in cancer metastasis. We showed that EMT and non-EMT cells cooperate to complete the spontaneous metastasis process. We thus hypothesize that EMT cells are responsible for degrading the surrounding matrix to lead the way of invasion and intravasation. Non-EMT cells then enter the blood stream and reestablish colonies in the secondary sites.

Figure 1

p12 antisense inhibits TGF-β-induced EMT on HaCaT cells. A, Phase-contrast images of p12 antisense transfectants of HaCaT cells. Zn-inducible p12 antisense transfectants (ip12(−)HaCaT) and vector control transfectants (pMTCB6⁺-HaCaT) were cultured in DMEM + 5% FBS in the presence of 25 μM ZnSO₄, and treated with or without a mixture of TGF-β (10 ng/ml) and EGF (30 ng/ml) for 48 h. B, Western blotting analysis of the E-cadherin and p12 protein levels in vector and p12 antisense transfectants with β-actin as a loading control.

Figure 2

p12 induces EMT in HCPC-1 cells. A, Phase-contrast images of vector and p12 transfectants. p12 expression altered cell morphology from polygonal, epithelial structure (left) to spindle-like, fibroblastoid (middle) structure. Mixed culture of p12 and vector transfectants segregated from each other (right). B, Fluorescent images of the mixed culture of vector and p12 transfectants shown in A, right panel. Red and green fluorescence generated from DsRed and GFP, respectively, represented vector and p12 transfectants. C, qRT-PCR analysis of E-cadherin mRNA levels in vector and p12 transfectants and in p12 transfectants treated with 5-Aza-cdR (left). Western blotting analysis of the desmoplakin, N-cadherin, E-cadherin and vimentin protein levels in HCPC-1 parental cells and in vector and p12 transfectants with β-actin as a loading control (right). D, IF of E-cadherin (green) and vimentin (red) in vector and p12 transfectants.

Figure 3

Dco-1 induces the expression of Twist2 that mediates EMT. A, RT-PCR analysis of known E-cadherin repressors. Twist2 expression was induced by p12. B, Western blotting analysis of Twist2 protein in vector and p12 transfectants. C, IF of Twist 2 protein in vector and p12 transfectants. Cell nuclei were stained with DAPI. Twists were detected both in the cytoplasm and in the nucleus (arrows). D, Knocking down Twist 2 reverted cell morphology to epithelial like structures (arrows). Twist2-specific synthetic siRNA and a plasmid encoding GFP were co-transfected into p12 transfectants (GFP free). Twist-2 siRNA-transfected cells are thus labeled with GFP expression.

Figure 4

p12-induced EMT enhances cell motility and local tumor invasion. A, Migration of vector and p12 transfectants from wounded edges in an in vitro cell migration assay. B, Average distances of cell migration in 24 h. A total of 100 cells were measured under a phase contrast microscope. C, H&E staining showed local invasion of the primary tumors derived from p12 transfectants (EMT) but not from vector transfectants (non-EMT). Tumor cells were indicated by stars. Double IF of tumor cells (indicated by stars) and blood vessels (indicated by arrows) showed intravasation of tumor cells only in the tumor tissues derived from p12 transfectants. Macro photos of the lungs and H&E examinations did not detect metastasis in the lung in all the animals in both groups (n=12). D, PCR analysis of GFP and DsRed genomic DNA collected from blood samples of the tumor-bearing mice.

Figure 5

Ability of non-EMT, EMT, and MET cells to establish lung metastasis from intravenous inoculation. A, All the animals (n=6) developed lung metastases from intravenous inoculation of vector transfectants (non-EMT cells) as shown by macro photos and H&E staining at both low and high magnifications. None of the animals developed lung metastasis from intravenous inoculation of p12 transfectants (EMT cells) and E-cadherin re-expressed p12 transfectants (MET cells). B, Western blotting analysis of E-cadherin confirmed E-cadherin re-expression. C, E-cadherin re-expression reverted the fibroblastoid structures of p12 transfectants to epithelial structures, suggesting an MET process.

Figure 6

A mixture of EMT and non-EMT cells completed the entire spontaneous metastasis process. A, Primary tumor tissues. H&E staining of the tumor edge showed invasive phenotype. Double IF of GFP and DsRed showed that the invasion front is composed of mainly p12 transfectants (green) but the tumor center consists of mainly vector transfectants (red). B, PCR detection of DsRed and GFP DNA from the blood samples and lung metastases. Both GFP and DsRed were detected in the blood stream but only DsRed was detectable in the metastatic foci in the lung. C, Lung metastases shown by macro photos and H&E staining. D, Fluorescent images and IHC staining of DsRed, GFP, and E-cadherin in the lung metastases. GFP was not detectable but both DsRed and E-cadherin are strongly expressed, indicating they were originated from vector transfectants (non-EMT cells).