Major Activities/Specific Objectives. The principal goal of our ongoing research effort is to develop an in depth mechanistic understanding of the MMP-independent activities of members of the TIMP family, in particular TIMP-2. We have identified the following specific objectives to obtain our goals: 1) examine the role of TIMPs in altering the growth and invasive potential of cancer stem cells (CSCs) in vitro;2) study to effects of TIMPs on primary and metastatic tumor growth in vivo;3) study the influence of TIMPs on recruitment of immune-modulatory cells (myeloid-derived suppressor cells (MDSC)) to the primary tumor and metastatic niche. A major focus in my lab has been to demonstrate the contribution of the MMP-independent anti-angiogenic effects 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 non-small cell lung carcinoma (NSCLC) 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 associated 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 glioblastoma, and suppresses angiogenesis, cell proliferation and VEGF-A expression. However, these findings need to be confirmed and the mechanisms of the effects on downstream gene regulation remain to be identified. Key outcomes and achievements. Additional data from our gene expression profiling also revealed changes in ATP-binding cassette (ABC) transporter gene expression in NSCLC A549. This has led to a new avenue of investigation directed at understanding the effects of TIMP-2 on cancer stem cells (CSC) and potential use of TIMP-2 to enhance cytotoxic therapies. 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 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 the biological activities of TIMP-2 that suppress growth of cancer stem cells, tumor cells, and angiogenesis, as well as tumor growth and metastasis in vivo, and provide a biologic basis for the potential use of TIMP-2 therapy in combination with chemo-and/or radiation therapy to enhance the efficacy of these treatments while reducing potential side effects. Angiogenesis and inflammation are important therapeutic targets in NSCLC. We examined the effects of TIMP-2 on NSCLC tumor-associated angiogenesis and inflammation were examined in TIMP-2-deficient mice and compared with wild type mice using the murine Lewis lung (LL) carcinoma model. TIMP-2-deficient mice demonstrated an increased growth of tumor, significantly elevated levels of vascular endothelial growth factor-A (VEGF-A) and enhanced expression of angiogenic markers in tumor tissues. In addition Tumor-bearing TIMP-2-deficient mice demonstrated up regulation of inflammatory mediators, nuclear factor-kappa B and Annexin A1, elevated serum levels of interleukin-6, and a significant increase of tumor infiltrating inflammatory cells. Phenotypic analysis revealed an increase of splenic MDSC (CD11b+ and Gr-1+ cells) expressing VEGFR-1 suggesting a systemic effect of TIMP-2 deficiency induced by the LL tumors. In contrast, forced overexpression of TIMP-2 in a human xenograft model of NSCLC using A549 cells demonsted a significant reduction in recruitment of MDSC to the tumors. The recruitment of MDSC to tumors has been linked to suppression of antitumor immunity and enhanced tumor angiogenesis. These recent findings suggest that TIMP-2 has a variety of effects on both tumor and host cells that combine to produce a potent anti-tumor activity that could be exploited clinically.
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