During the past 4 years we have completed, (and published) several studies characterizing downstream signaling pathways from the TIMP-2-receptor, alpha3/beta1-integrin, involved in mediating the anti-angiogenic effects of TIMP-2, including a more detailed characterization of TIMP-2-mediated growth inhibition of endothelial cells (hMVECs) in response to fibroblast growth factor-2 (FGF-2) These studies demonstrated that inhibition of src homology protein tyrosine phosphatase-1 (SHP-1), either by pharmacologic methods or expression of dominant negative SHP-1, prevents TIMP-2-mediated inhibition of fibroblast growth factor mediated endothelial mitogenesis. These findings are consistent with previous observations that TIMP-2 activates SHP-1 resulting in inactivation (dephosphorylation) of a variety of receptor tyrosine kinases via a ligand-dependent fashion In another series of experiments, we examined the phosphorylation pattern of vascular endothelial growth factor-2 (VEGFR-2) following VEGF-A stimulation in the presence or absence of TIMP-2 (Ala+TIMP-2) Our results show that Ala+TIMP-2 selectively alters tyrosine phosphorylation of VEGFR-2 at residues implicated in endothelial cell proliferation and migration (significantly decreased phosphorylation at Y951, Y996 and Y1175). Ala+TIMP-2 disrupted downstream activation of phospholipase C-gamma, Akt and endothelial nitric oxide synthetase. TIMP-2 or Ala+TIMP-2 inhibit VEGF-A-mediated Ca+2 influx, and reduced cGMP levels normally enhanced by nitric oxide donors. The observed decrease in cGMP was sensitive to isobutylmethylxanthine inhibition. In another recent report we showed that TIMP-2 mediates the inhibition of vascular permeability via an alpha3/beta1-Shp-1-cAMP/PKA signaling pathway, which enhanced VE-cadherin association with the cytoskeleton Our results demonstrate the potential utility for TIMP-2 in cancer therapy through """"""""normalization"""""""" of vascular permeability and function . It is this """"""""normalization"""""""" of tumor vasculature, to which Jain and colleagues attribute much of the clinical benefit and enhanced response to radiotherapy following the use of angiogenesis inhibitors It is interesting that such """"""""normalization"""""""" also seems to occur in vivo in our experiments utilizing forced expression of TIMP-2 in tumor xenografts (major unpublished observation, see below). We also completed our studies on the """"""""B-C loop"""""""" synthetic peptides, identified by their alpha3/beta1-binding activity, demonstrating their anti-angiogenic and anti-tumorigenic activity in vivo. The proposed TIMP-2 mutants of the glutamic, lysine and arginine amino acid residues in the """"""""B-C"""""""" loop region have been expressed but their activities have not yet been further characterized. As noted above these studies are incomplete because of staff departures and our focus on demonstrating in vivo MMP-independent activity of TIMP-2. As mentioned above our major focus has been to demonstrate the MMP-independent anti-angiogenic effects of TIMP-2 in contributing to the anti-tumor activity of TIMP-2 in vivo observed by a number of investigators. To this end we employed retroviral vectors to force expression of TIMP-2 and Ala+TIMP-2 in the human lung carcinoma cell line A549 and then used these cell lines in tumor xenograft experiments in both nu/nu and NOD-SCID mice. Although these cell lines showed no discernable difference in basal growth rates in vitro there was significant suppression of tumor growth in both TIMP-2 (90 %) and Ala+TIMP-2 (75%) xenografts compared to empty vector controls as late as 40 days post tumor-inoculation. The suppression of tumor growth was accompanied by a statistically significant decrease in tumor microvascular density count (CD 31+ or CD34+), a measure of antiangiogenic effects, as well as by increased tumor cell apoptosis (also possibly due to inhibition of angiogenesis). Somewhat unexpectedly, we also observed a decrease in focal adhesion kinase (FAK) in TIMP-2 expressing tumors and a significant decrease in FAK phosphorylation (Y397) in both TIMP-2 and Ala+TIMP-2 expressing tumor cells. Our observation that both FAK and/or AKT (Protein Kinase B, PKB) phosphorylation is reduced in TIMP-2 and Ala+TIMP-2 tumor tissues is significant in that: 1) FAK is upstream of AKT signaling, and both are involved in regulation of cell migration;2) TIMP-2 and Ala+TIMP-2 expression reduced tumor cell migration in vitro. We previously reported decreased FAK phosphorylation in endothelial cells where it is involved in control of eNOS activity. In summary, these experiments using retrovirally transduced tumor cells expressing wild type (wt) TIMP-2 or metalloprotease inhibitor-deficient Ala+TIMP-2 clearly demonstrate that the MMP-independent activities of TIMP-2, including the anti-angiogenic activity, are of sufficient magnitude to significantly impact tumor growth in vivo. Our observation of the effects of TIMP-2 and Ala+TIMP-2 on A549 tumor xenografts, led us to perform transcriptional profiling of these cell lines and tumor tissues. The observed changes in gene expression are predominantly related to decreased tumor development and reduced metastasis In contrast to control A549 cells, cells expressing TIMP-2 or Ala+TIMP-2 showed increased expression of E-cadherin, and were resistant to redistribution of cell membrane associate E-cadherin and beta-catenin following epidermal growth factor (EGF) stimulation, suggestive of a mesenchymal-epithelial transition. Other genes of interest that were differentially regulated include EGF-containing fibulin-like extracellular matrix protein 1 (EGFEMP1, fibulin 3) that was up regulated in cells expressing TIMP-2 or Ala+TIMP-2. This protein is a favorable prognostic factor in gliomablastoma, and suppresses angiogenesis, cell proliferation and VEGF-A expression. However, these findings need to be confirmed (see below) and the mechanisms of the effects on downstream gene regulation remain to be identified. Additional data from our gene expression profiling also revealed changes in ATP-binding cassette (ABC) transporter gene expression. ABC proteins drive cell efflux of a variety of substrates, including cytotoxic drugs, and are known to contribute to resistance to cancer chemotherapy. The activity of ABC transporters is an important indicator of cancer stem cell (CSC) presence in various solid tumors. The Hoechst 33342 dye efflux assay identifies a tumor cell subpopulation, known as the side population (SP), that is enriched in CSCs. Based on our gene expression profiling data we posit that TIMP-2 anti-tumor activity may, in part, involve regulation of the SP in our lung cancer cell model. To this end, we determined the correlation between the SP fraction and level of endogenous TIMP-2 expression in a series of six non-small cell lung cancer (NSCLC) cell line. Interestingly, our results demonstrate a strong, highly significant inverse correlation (R2=0.073, p0.03) between the level of endogenous TIMP-2 mRNA expression and the percentage of SP determined using the Hoechst dye efflux assay. In A549 cells expressing TIMP-2, a significant decrease in the SP is observed and this decrease is associated with lower expression of ABCG2, ABCB1 and AKR1C1. Functional analysis reveals that A549 cells expressing TIMP-2 show increased sensitivity to cytotoxic drugs, including doxorubicin and topotecan. These findings suggest that TIMP-2 therapy may enhance sensitivity to cytotoxic chemotherapy, and are the first demonstration that TIMP-2 modulates SP and possibly CSC levels and function. We feel these studies demonstrate significant progress in developing a new biological activity that suppresses cancer stem cells, tumor cells, endothelial cells and tumor growth in vivo.
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