March 2006
Volume 5
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March 2006
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Contents
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Studying Tumor-Host Interactions Reveals a Novel Mechanism for the Activity of TIMP-2
The model is based on altering the expression of a single factor by the tumor cell using retroviral transduction and studying the effects of this change on the surrounding host environment and on the tumor cells themselves growing in vivo. The effects seen in vivo can then be compared to the differences seen between the altered cell line and the wild-type parental cell line in vitro; those changes unique to the in vivo observations can be ascribed to a relationship between the tumor and the host. To test this model system, we chose to study tissue inhibitor of metalloproteinase 2 (TIMP-2). TIMP-2 is an endogenous protein present in a variety of tissues and characterized by its ability to both block metalloproteinase activation in the extracellular matrix and to inhibit the development of blood vessels. Previous work has demonstrated that these two activities may be attributable to separate domains of the TIMP-2 protein (i.e., truncated forms of the protein have different activities, with one portion inhibiting metalloproteinase activity and an alternative portion inhibiting angiogenesis). To identify the pathways through which TIMP-2 mediates its antiangiogenic activity in vivo, we applied the following experimental design. A murine colon cancer line, MC38, was chosen because of its ability to form significant tumor neovasculature when grown as subcutaneous tumors in syngeneic BL/6 mice. The TIMP-2 gene was cloned into a retroviral vector, and MC38 cells were transduced with either a TIMP-2–expressing retrovirus or a null retrovirus control. Clones were selected, and a high-expressing TIMP-2 clone was chosen for further study, which was identified as MET-11. MET-11 and the null retrovirus–transduced tumor line, MEX, demonstrated no difference in their in vitro growth characteristics. MET-11 and MEX cells were then injected subcutaneously into BL/6 mice and were allowed to grow for 18 days. Between day 6 and day 18, MET-11 tumors were significantly smaller then their MEX or wild-type counterparts and had significantly less vascularity as determined by immunohistochemical staining of the tumors with CD31 antibody and vessel counts. This observation was consistent with the known antiangiogenic activity of TIMP-2. Tumors were harvested at day 6 and day 12. RNA was extracted from both MET-11 and MEX tumors, and cDNA microarray analysis was performed. A comparison was also made between MET-11 and MEX cells grown in vitro. Figure 1 depicts the array analysis schema. Figure 1. Identification of genes associated with the host response to tissue inhibitor of metalloproteinase 2 (TIMP-2). A) Strategy for comparing gene expression patterns of MC38/null and MC38/TIMP-2 tumor cells in vitro and in vivo. Using cDNA microarrays, MC38/null (green) and MC38 TIMP-2 (red) were compared in vitro and in vivo after 6 or 12 days of growth. Genes associated with tumor-host interactions due to TIMP-2 might be similarly expressed in vitro (e.g., yellow spot in lower left corner of top array, arrowhead), but differentially expressed in vivo (red spot in lower left corner of bottom two arrays, arrowheads). B) cDNA microarray analysis identified 13 such genes. Each pixel represents the expression ratio on one array. Red indicates upregulation in the MC38/TIMP-2 sample, and green indicates downregulation. Color intensity is proportional to expression ratio. Black represents ratios close to 1.0. C) Microarray and qRT-PCR data for PTPN16, the murine gene for mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1). Expression ratios were close to 1.0 in vitro, but showed upregulation in MC38/TIMP-2 tumors in vivo. To identify differentially expressed genes between MET-11 and MEX tumors grown in vivo, we chose day 6 for analysis, as this was a time point in which both MET-11 and MEX tumors were of similar size. We hypothesized that the gene differences seen here have a cause-effect relationship on the change in growth characteristics seen between day 6 and day 18. Gene expression changes were also compared at day 12 to look for those genes that were persistently altered in expression between MET-11 and MEX tumors. Those genes that were altered between MET-11 and MEX at day 6 and persisted through day 12 in vivo but were not altered in vitro were selected for further study. We found 13 genes to be differentially expressed between MET-11 and MEX tumors that fulfilled our criteria of greater than 2-fold up- or downregulation at both day 6 and day 12 in vivo and no differential expression in vitro. Among these genes PTPN16 (MKP1) was found to be upregulated to the greatest degree in MET-11 tumors compared to MEX tumors at day 6. PTPN16 is a protein-tyrosine-phosphatase that dephosphorylates p38 MAP kinase, thus inactivating it. p38 MAP kinase is known to play an important role in both vascular endothelial grow factor (VEGF) and basic fibroblast growth factor (bFGF) signaling, and therefore, its modulation may be important with respect to TIMP-2’s angiogenic inhibitory activity. We sectioned MET-11 and MEX tumors at day 6 and analyzed the levels of protein expression for PTPN16, p38 MAP kinase, and phosphorylated p38 MAP kinase. We found, in concordance with the RNA data, that PTPN16 expression was significantly elevated in TIMP-2–expressing tumors (MET-11) compared with their null-transduced counterparts (MEX). In addition, whereas total p38 levels were similar in both tumors, the proportion of phosphorylated p38 was significantly reduced in the MET-11 TIMP-2 overexpressers. This observation fit with the increased levels of PTPN16. To test whether increased expression of PTPN16 and therefore decreased phosphorylation of p38 led to the impaired growth we saw in TIMP-2 overexpressing tumors, we inoculated BL/6 mice with 1 × 106 MET-11 tumor cells in their flank. Tumors were allowed to grow for 14 days, at which time mice were divided into two groups: one group received systemic phosphate-buffered saline (PBS) injections from day 14 until day 25, whereas the other group received systemic injections of orthovanadate (a phosphatase inhibitor) over the same time period. Tumors growing in the mice receiving orthovanadate grew significantly larger than did those in the mice receiving the PBS control. Tumors harvested from mice receiving orthovanadate compared with tumors harvested from mice receiving PBS showed increased phosphorylation of p38 MAP kinase consistent with an inhibition of PTPN16 activity. This model system, which allowed us to study tumor host-interactions, led us to hypothesize a new mechanism of action for TIMP-2 with respect to its effects on tumor blood vessel growth. TIMP-2 upregulates the expression of PTPN16, resulting in a decrease in the phosphorylation status of p38 MAP kinase. Inactivation of p38 MAP kinase inhibits the ability of VEGF and bFGF to signal through their receptors. Since VEGF and bFGF are important mitogens for endothelial cell proliferation, this inhibition would be expected to impair the ability of a tumor to develop a blood supply. This model system can be used to study other genes to identify their in vivo mechanisms of action and represents a technique for applying both genomic and proteomic approaches to the study of tumor-host interactions.
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umor
growth, invasion, and metastasis are the results of a complex series of interactions
between tumor cells and the cells that make up the host microenvironment. Each
of the cell types involved in this process has the potential to influence the
other cell types through secreted cytokines and through alterations of the environment,
such as changes in pH and oxygen content. This complex interplay is extremely
difficult to model in an in vitro system. This difficulty led us to develop
an in vivo model system that would allow these interactions to be studied
at both a genomic and proteomic level. 