TSP-1 inhibits activated fibroblasts from invading tumor cell cluster

tumor cell clusters (Ishii G et al. 2005). Since TSP-1 directly reduced MMP-2 activity and TGF-β-induced migration ability, we examined the effect of TSP-1 on invasive ability of activated fibroblasts into tumor cell cluster. NIH3T3 cells but not human normal fibroblasts (NF), expressed α-SMA and had the ability to invade SiHa tumor cluster, regardless of the expression status of TSP-1 in tumor cells (Figure 7, A). Exogenous TSP-1 (10, 20 or 40 μg/ml) in tumor cluster also failed to inhibit NIH3T3 from invading SiHa tumor cluster (Figure 7, B). By contrast, the invasive ability of NIH3T3-TSP-1 cells to SiHa (or SiHa-TSP-1) tumor cluster was

significantly reduced when compared with NIH3T3, indicating a direct inhibitory effect of TSP-1 on the invasive ability of NIH3T3 (Figure 7, A). We next examined the effect of exogenous TSP-1 on the invasive ability of NIH3T3 cells. Consistent with stable expression of TSP-1 in NIH3T3 cells, we found a dosage-dependent inhibition of NIH3T3 cell invasion. A complete inhibition was observed at 20 μg/ml or higher (Figure 7, C). We further used TGF-β to activate NIH3T3 and investigated the effect of TGF-β on TSP-1-mediated inhibition of NIH3T3 invasion.

TSP-1 potently inhibited fibroblast invasion regardless of the presence of TGF-β although a higher dose of TSP-1 was required for a complete inhibition of TGF-beta-treated NIH3T3 cells (Figure 7, C and D). Taken together, our results indicate that the TSP-1-mediated inhibition of the invasive ability could only be demonstrated when the manipulation of TSP-1 expression was in fibroblasts, but not tumor cells.

4.5 Discussion

Our current understanding on the role of TSP-1 in tumor progression and clinical prognosis has extended far beyond anti-angiogenesis. The present study provides evidence that TSP-1 has the potential to inhibit stroma reaction during cervical carcinogenesis. This conclusion is supported by the following findings: (i) The concordance of the downregulation of TSP-1 and the upregulation of stroma markers in surgical specimens of cervical carcinoma suggests that TSP-1 plays an inhibitory role in stroma reaction. (ii) Transfection of SiHa cells with a plasmid expressing the TSP-1 protein resulted in reduced tumor growth in SCID mice that was accompanied by a decrease in tumor vascularization and a lower level of stroma markers, α-SMA and desmin, than the vector transfection. (iii) Ectopic expression of TSP-1 or by addition of purified TSP-1 manifested an inhibition of MMP-2 activity, TGF-β-enhanced cell migration, and the invasive ability of activated fibroblasts from tumor cell clusters.

We previously pointed out that the switch of angiogenic phenotype, partly due to the down-regulation of TSP-1, occurred during the transition from low-grade to high-grade squamous intra-epithelial lesion (Wu MP et al. 2004). In this study, we demonstrated a temporal inverse correlation of TSP-1 and stromal marker expression during cervical carcinogenesis using human clinical specimens. The inhibitory effect of TSP-1 on stromal marker expression was further confirmed in SCID mouse xenografts using transfection of TSP-1 cDNA expression vectors.

Genetic manipulation of TSP-1 expression level in the cells demonstrated that TSP-1-mediated inhibition of stroma reaction was primarily due to the inhibition of activated fibroblast migration and invasion, rather than a direct effect on the stromal maker expression. These results indicate that TSP-1 participates not only in the negative regulation of angiogenesis but also stroma reaction during cervical

carcinogenesis.

Cancer progression is a complex process involving transformation, invasion, angiogenesis and metastasis (Hanahan D and Weinberg RA 2000). Although TSP-1 is commonly believed to have anti-tumor effects due to its anti-angiogenic ability, however, results from various studies have demonstrated different correlations among the levels of TSP-1 and tumor progression and clinical prognosis in different tumor types, indicating different biological functions of TSP-1 in different cancer cell types. The lower expression levels of TSP-1 in cervical cancer than those in normal tissue is similar to some human tumors in which decreased TSP-1 expression is associated with malignancy (Bunone G et al. 1999;

de Fraipont F et al. 2000). Among these cancers, TSP-1 expression is inversely correlated with tumor grade and survival rate in thyroid, colon and bladder carcinomas (Bunone G et al. 1999; de Fraipont F et al. 2001). The prognostic value of TSP-1 in cervical cancer requires further investigation. In contrast, TSP-1 expression was higher in tumors or tumor-associated stroma than in normal epithelial or stroma tissue in other cancer types (Bertin N et al. 1997). Thus, TSP-1 seems to have activatory as well as inhibitory properties in tumor progression.

There are several likely explanations to account for its dualistic effects. Firstly, TSP-1 interacts with multiple extracellular macromolecules and cell surface receptors, thus exerting a wide range of responses (Brown EJ and Frazier WA 2001; de Fraipont F et al. 2001). Secondly, the exposure to high stromal TSP-1 may induce expression of angiogenesis activators in tumor cells, which override the effects of TSP-1 (Fontana A et al. 2005). Thirdly, TSP-1 exerts its effects on multiple types of stromal cells such as inhibiting fibroblasts migration (Streit M et al.

2000), decreasing the recruitment of inflammatory cells (Doyen V et al. 2003), inducing apoptosis of endothelial cells (Reiher FK et al. 2002; Streit M et al. 1999),

or activating smooth muscle cells proliferation (Isenberg JS et al. 2005a). Fourthly, some limitations still remain among the various models (Hlatky L et al. 2002).

It has become increasing clear that, from the context of tumor-stroma interactions, stroma plays an active role in tumor progression (Mueller MM and Fusenig NE 2004). Stromal cells can acquire oncogenic transformation following the exposure to carcinogen (Kalas W et al. 2005), manipulation of MMPs (Rodriguez-Manzaneque JC et al. 2001), and the recruitment of inflammatory cells to the stroma (Doyen V et al. 2003; Vallejo AN et al. 2000). Stroma reaction is often accompanied with stromal marker expression and functional changes into an invasive phenotype. Inhibition of stroma reaction by TSP-1 might be through reduced expression of stromal markers and invasiveness or inhibition of activated fibroblast migration and recruitment to tumor stroma. Using Western blot analysis, migration and Matrigel co-culture invasion assays, we demonstrated that TSP-1 potently exerted inhibitory effect on the migration and invasion of fibroblasts with or without TGF-β treatment, however, it has little effect on the expression level of α-SMA expression, a typical marker for activated fibroblasts. Unlike TSP-1, secreted protein, acidic and rich in cysteine (SPARC) was shown to inhibit fibroblast activation by blocking α-SMA overexpression (Chlenski A et al. 2007b).

Although SPARC and TSP-1 are both matricellular proteins that inhibit angiogenesis and interfere with the organization of the extracelluar matrix;

however, TSP-1 inhibits stroma reaction through a mechanism distinct from SPARC.

It is worth mentioning that the stroma reaction occurred mainly at the tumor-stroma junction as shown in both clinical and animal specimens, this phenomenon was attenuated by the upregulation of TSP-1. Furthermore, this highlights the importance of the interaction between tumor and stroma, and

suggests a likely possibility that modulation of tumor microenvironment can potentially change the cell-matrix interactions associated with cell movement and further progression. Consistent with this notion, it has been shown that TSP-1 inhibits the activity of MMP-2 and MMP-9 via a direct interaction with these proteases in specific regions (Bein K and Simons M 2000; Rodriguez-Manzaneque JC et al. 2001). In this study, no MMP-9 activity could be detected in NIH3T3 cells.

Instead, we observed that TSP-1 inhibited MMP-2 activity, which was not altered by TGF-β treatment, followed by a decrease in the migration and invasive ability of activated fibroblasts. More studies are needed to address the exact mechanism whereby TSP-1 decreased MMP-2 activity in the context of activated fibroblasts.

Our present results show that TSP-1 inhibited TGF-β-enhanced migration and the invasive ability of NIH3T3. TSP-1 is known to function as an activator of TGF-β and this activity is mapped to the type-1 repeats of TSP-1 (TSRs) (Crawford SE et al. 1998; Kawataki T et al. 2000). Moreover, the activation of TGF-β by TSP-1 can either inhibit (Miyanaga K et al. 2002) or increase (Kawataki T et al. 2000) malignancy depending on cell types. The complexity and duality in the functions of TSP-1 and TGF-β may be due to their ability to suppress tumor cell proliferation at early stages, but to enhance tumor invasion and metastasis by enhancing the host stroma reaction at later stages (Radisky DC and Bissell MJ 2004). Further investigation is needed to dissect the dynamic interaction between TSP-1 and TGF-β in the regulation of cervical cancer growth.

Furthermore, TSP-1 expression in both the stroma and tumor areas of SCID mouse xenografts were different from that mainly expressed in basal epithelia of normal human cervix. This discrepancy may stem from species differences, differential sensitivities of antibodies to murine and human samples, host compensatory response or different study model (heterotopic versus orthotopic

animal model). In addition, clinical observations and genetic manipulations in our study have pointed to TSP-1 as a central mediator to inhibit the invasive ability of activated fibroblasts and their subsequent recruitment to stroma. However, we cannot completely rule out the possibility of other factors, e.g. the interaction with TGF-β, involved in stroma reaction (Hugo C 2003).

To summarize, our current study demonstrates that TSP-1 plays a broader role in tumor cell biology including the inhibition of activated fibroblast migration and invasion, in addition to its well-known anti-angiogenic effects. In viewing the fact that stromal therapy has recently emerged as a strategy for cancer treatment (Meyerhardt JA and Mayer RJ 2005), this study carries significant implications with regard to the application of targeted interventions on the TSP-1-mediated stroma reaction may represent a potentially new strategy for inhibiting the progression of cancer.

Acknowledgements:

This work was supported by grants, 93-CM-TMU-02, 94CM-KMU-03 and 95CM-TMU-03 from Chi Mei Foundation Hospital, and Center of Excellence for Clinical Trial and Research in Oncology Specialty, Ministry of Health, Taiwan. We thank Professor Tien Kuo, Department of Molecular Pathology, MD Anderson Cancer Center for critical reading of this manuscript, and Miss Chin-Li Lu, Chi Mei Foundation Hospital, for statistic analysis assistance.