Balanced expression of proteases and their inhibitors is one prerequisite of tissue homeostasis. Metastatic spread of tumor cells through the organism depends on proteolytic activity and is the death determinant for cancer patients. Paradoxically, increased expression of tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of several endometalloproteinases, including matrix metalloproteinases and a disintegrin and metalloproteinase-10 (ADAM-10), in cancer patients is negatively correlated with their survival, although TIMP-1 itself inhibits invasion of some tumor cells. Here, we show that elevated stromal expression of TIMP-1 promotes liver metastasis in two independent tumor models by inducing the hepatocyte growth factor (HGF) signaling pathway and expression of several metastasis-associated genes, including HGF and HGF-activating proteases, in the liver. We also found in an in vitro assay that suppression of ADAM-10 is in principle able to prevent shedding of cMet, which may be one explanation for the increase of cell-associated HGF receptor cMet in livers with elevated TIMP-1. Similar TIMP-1-associated changes in gene expression were detected in livers of patients with metastatic colorectal cancer. The newly identified role of TIMP-1 to create a prometastatic niche may also explain the TIMP-1 paradoxon. [Cancer Res 2007;67(18):8615-23]
Matrix metalloproteinases (MMPs), and in particular gelatinases (MMP-2 and MMP-9), play a key role in cancer progression. However, clinical trials in which MMP inhibitors were tested in cancer patients have been disappointing. Whereas many reasons have been postulated to explain the failure of the clinical trials, lack of inhibitor selectivity was a major limitation. Thus, despite the consensus opinion that MMP-mediated proteolysis is essential for cancer progression and that certain MMPs represent important targets for intervention, effective and selective inhibition of those MMPs remains a major challenge in drug development. We previously reported the first mechanism-based MMP inhibitor, designated SB-3CT, which is a selective gelatinase inhibitor. Here we report that SB-3CT (5-50 mg/kg/d) is a potent inhibitor of liver metastasis and increases survival in an aggressive mouse model of T-cell lymphoma. This study shows that mechanism-based inhibition of gelatinases represents a novel approach to inhibitor design that promises to be a successful anticancer therapy. (Cancer Res 2005; 65(9): 3523-6)
The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP-(diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.ADP-ribosylation of eEF2 | Pseudomonas exotoxin | diphtheria toxin | translation | DPH gene knockout
In many different tumor entities, increased expression of tissue inhibitor of metalloproteinases-1 (Timp-1) is associated with poor prognosis. We previously reported in mouse models that elevated systemic levels of Timp-1 induce a gene expression signature in the liver microenvironment increasing the susceptibility of this organ to tumor cells. This host effect was dependent on increased activity of the hepatocyte growth factor (Hgf)/hepatocyte growth factor receptor (Met) signaling pathway. In a recent study we showed that Met signaling is regulated by Timp-1 as it inhibits the Met sheddase A disintegrin and metalloproteinase-10 (Adam-10). The aim of the present study was to elucidate whether the metastatic potential of tumor cells benefits from autocrine Timp-1 as well and involves Adam-10 and Met signaling. In a syngeneic murine model of experimental liver metastasis Timp-1 expression and Met signaling were localized within metastatic colonies and expressed by tumor cells. Knock down of tumor cell Timp-1 suppressed Met signaling in metastases and inhibited metastasis formation and tumor cell-scattering in the liver. In vitro, knock down of tumor cell Timp-1 prevented Hgf-induced Met phosphorylation. Consequently, knock down of Met sheddase Adam-10 triggered auto-phosphorylation and responsiveness to Hgf. Accordingly, Adam-10 knock down increased Met phosphorylation in metastatic foci and induced tumor cell scattering into the surrounding liver parenchyma. In conclusion, these findings show that tumor cell-derived Timp-1 acts as a positive regulator of the metastatic potential and support the concept that proteases and their natural inhibitors, as members of the protease web, are major players of signaling during normal homeostasis and disease.
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