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. 2011 Nov 15;10(22):3834-40.
doi: 10.4161/cc.10.22.18294. Epub 2011 Nov 15.

MET-dependent Cancer Invasion May Be Preprogrammed by Early Alterations of p53-regulated Feedforward Loop and Triggered by Stromal Cell-Derived HGF

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Free PMC article

MET-dependent Cancer Invasion May Be Preprogrammed by Early Alterations of p53-regulated Feedforward Loop and Triggered by Stromal Cell-Derived HGF

Chang-Il Hwang et al. Cell Cycle. .
Free PMC article

Abstract

MET, a receptor protein tyrosine kinase activated by hepatocyte growth factor (HGF), is a crucial determinant of metastatic progression. Recently, we have identified p53 as an important regulator of MET-dependent cell motility and invasion. This regulation occurs via feedforward loop suppressing MET expression by miR-34-dependent and -independent mechanisms. Here, by using Dicer conditional knockout, we provide further evidence for microRNA-independent MET regulation by p53. Furthermore, we show that while MET levels increase immediately after p53 inactivation, mutant cells do not contain active phosphorylated MET and remain non-invasive for a long latency period at contrary to cell culture observations. Evaluation of mouse models of ovarian and prostate carcinogenesis indicates that formation of desmoplastic stroma, associated production of HGF by stromal cells and coinciding MET phosphorylation precede cancer invasion. Thus, initiation mutation of p53 is sufficient for preprogramming motile and invasive properties of epithelial cells, but the stromal reaction may represent a critical step for their manifestation during cancer progression.

Figures

Figure 1
Figure 1
miR-34-independent regulation of MET in microRNA-free environment. (A) Met expression in primary OSE cells isolated from Dicerfl/fl, p53fl/fl and Dicerfl/flp53fl/fl mice. Met mRNA expression (mean ± SD, n = 3) was measured by qRT-PCR 48 h after infection with Ad-Cre or Ad-LacZ. (B) MET expression in p53-/-Dicer-/- OSE cells 48 h after transfection with either empty vector (pORF) or pORF-hp53.
Figure 2
Figure 2
Expression of phosphorylated MET (P-MET) during OSE carcinogenesis. Normal OSE (Control, arrow) and preinvasive neoplastic cells (60 d, arrow) above the regular stroma express negligible levels of P-MET. Preinvasive OSE (170 d, arrow) above desmoplastic stroma (arrowhead) and invasive neoplastic OSE cells (270 d, arrow) contain detectable amount of P-MET. Note expression of HGFα (HGF) and F4/80 in macrophages (60 d, arrowheads) and desmoplastic cells and macrophages/histiocytes (170 and 270 d, arrowheads). Hematoxylin-eosin (H&E) and ABC Elite method, hematoxylin counterstaining (P-MET, HGF and F4/80). Calibration bar, all images, 50 µm.
Figure 3
Figure 3
Hgf expression in OSE cells and fibroblasts after p53, Rb or p53/Rb inactivation. Signal intensity of Hgf expression (mean ± SD, n = 3) was measured by Affymetrix GCOS software and normalized by scaling each GeneChip to a target signal of 500. **p < 0.01.
Figure 4
Figure 4
Expression (arrows) of total (MET) and phosphorylated (P-MET) MET and HFGα (HGF) in prostate neoplastic lesions. Carcinomas (Ca) and prostate intraepithelial neoplasms (PIN), are located, respectively, in the proximal and distal regions of prostatic ducts of mice with prostate epithelium-specific inactivation of p53 and Rb. Note absence of MET expression in PIN and HGFα location in stromal (arrow) but not epithelial (arrowhead) cells. ABC Elite method, hematoxylin counterstaining. Calibration bar, 50 µm.
Figure 5
Figure 5
Proposed model of MET-dependent cancer invasion preprogrammed by early alterations of p53-regulated feedforward loop and triggered by stromal cell-derived HGF. Feedforward loop regulation of MET by wild-type p53 consists of miR-34-dependent and -independent mechanisms. p53 protein with point mutations in its p53 DNA binding domain is unable to transactivate MET targeting miR-34. However, it still binds SP1, thereby repressing MET promoter, albeit to a limited extent. In contrast, lack of p53 due to null mutations results in incapacitations of both miR-34 transactivation and MET promoter repression, thereby leading to highest levels of MET. Although p53 mutations result in increased expression of MET, cells become highly motile and invasive only after MET activation by phosphorylation (P-MET). This phosphorylation is triggered by MET ligand HGF produced by cells of newly forming desmoplastic stroma. Thus, while alterations in p53/miR-34/MET network preprogram cells for increased motility and invasion, microenvironment may play a crucial role in triggering those properties during cancer progression.

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