Current literature provides evidence of an association between hypoxic conditions and the difficulties of treating brain tumors, like glioma. Hypoxia has been implicated in many aspects of tumor development, angiogenesis and growth . At the cellular level, hypoxia induces the expression and cellular concentration of HIF-1α. High expression of this factor leads to an increase in cell division-tumorigenesis and appears to be a prognostic marker for malignancy [19, 20].
ADAMs comprise a family of proteins that contain both a disintegrin and a Zn-dependent metalloproteinase . These molecules are involved in gene regulation, cell adhesion and proteolysis. The most extensively studied protein belonging to this family is ADAM17 (a.k.a. TACE). ADAM17 sheds a variety of epidermal growth factors receptor (EGFR)-binding ligands, including transforming growth factor-alpha (TGF-α), heparin-binding epidermal growth factor (HB-EGF), and amphiregulin [6, 22]. In addition, ADAM17 facilitates the release of several integrins from the cell surface, and influences the invasive activity of several cells, including brain tumor [6, 21]. Sp1 is important to the transcription of many genes that contain GC boxes in their promoters . Sp1 has been widely perceived as a basal transcription factor since its discovery; however, increasing evidence suggests Sp1 regulates a multiple functions critical to tumorigenesis and progression [12, 14, 23]. Knowing that ADAM17 contributes to the invasiveness of tumor cells and that Sp1 binds to its promoter region, it is possible that Sp1 transcription factor may be a new target for anti-invasive therapies [14, 23].
Previously, we have reported that the increased invasion ability of U87 cells under hypoxic conditions is mediated by elevated ADAM17 expression and protease activity [6, 19]. Sp1 protein expression has been reported to increase in tumor cells under hypoxic conditions . We used the TESS promoter analysis program to determine if the Sp1 transcription factor binds to ADAM17, as the promoter region of ADAM17 contained multiple Sp1 transcription factor binding sites . Using a DNA-protein binding assay under normoxic conditions we found that Sp1 binds to ADAM17 within the ADAM17 promoter region, -901 to -804 of TSS. As one consensus sequence for human Sp1 is found at bp 3-9 of the ADAM17 promoter, we surmise this is the position of Sp1-binding; however mutational analysis is needed to confirm this is the target site. Sp1 down-regulation reduced expression of ADAM17 under both normal and hypoxic conditions; however, we have not confirmed the Sp1 binding site within the ADAM17 promoter is functional. Furthermore, it has been demonstrated that hypoxia can not only alter expression, but enhance the binding activity of Sp1 . Thus, although we demonstrate binding of Sp1 to the ADAM17 promoter, further investigation of its transcriptional effect upon ADAM17 is warranted.
Previous studies have shown that at the transcriptional level, Sp1 plays a critical role in gene expression especially under hypoxic conditions [12, 23, 25]. Our PCR data revealed that hypoxia induced mRNA expression of ADAM17 as well as Sp1. In addition, we observed that our Sp1-deficient cells decreased mRNA expression of ADAM17 under both normoxic and hypoxic conditions. Using Western blot, we confirmed that hypoxia induced protein expression of ADAM17 and Sp1. However, when Sp1 was down-regulated by an expression plasmid encoding for siRNA, hypoxia failed to induce ADAM17 mRNA and protein expression indicating that Sp1 is required for hypoxic-induction of ADAM17.
Previously, we have reported that increased ADAM17 expression and protease activity contributes to hypoxic-induced tumor invasion. In this study, we established that Sp1 regulates ADAM17 gene expression. Furthermore, we investigated whether inhibition of Sp1 would elicit an anti-invasion effect similar to inhibition of ADAM17. Here, we used an alpha-secretase assay to determine if Sp1 siRNA influences ADAM17 protease activity. As expected, hypoxia increased alpha-secretase activity in U87 tumor cells. However, when Sp1 was down-regulated, hypoxia did not significantly increase alpha-secretase activity in line with inhibition of hypoxia-induce ADAM17. Of note, Sp1 down-regulation did not decrease alpha-secretase activity under normoxic conditions. This is in agreement with our previous data that ADAM17 does not constitute for the majority of alpha-secretase activity in U87 cells under normoxic conditions, but does account for the majority of hypoxic-induced alpha-secretase activity .
ADAM17 mediates hypoxic-induced glioma invasion [5, 6, 26]. To test if Sp1 contributes to the invasion of tumor cells, we used an in vitro invasion assay. Our results indicate that under hypoxic conditions the invasive ability of U87 significantly increased, and this increase was correlated with high ADAM17 expression and proteolytic activity. The invasive ability of U87 cells decreased considerably when Sp1 was suppressed under both normoxic and hypoxic conditions. Similar to invasion, Sp1 down-regulation resulted in a significant reduction in U87 cell migration both under hypoxic and normoxic conditions.
Here we demonstrate that Sp1 is critical for hypoxic-induced ADAM17, and that Sp1 contributes to hypoxic induced glioma invasion. However, we have not established the effect of Sp1 upon invasion is solely mediated via ADAM17. In addition to many other genes, HIF-1α contains Sp1 binding sites in its promoter . In fact, we found Sp1 down-regulation diminished HIF-1α expression. Furthermore, the inhibitory effects of Sp1 down-regulation upon cell invasion and migration were more pronounced under hypoxic conditions, suggesting the role of Sp1 is more pronounced in the context of hypoxic-inducible factors. Hypoxic-induced ADAM17 expression is dependent upon Sp1, and ADAM17 significantly contributes to hypoxic-induced glioma invasion . However, it is probable the effect of Sp1 upon hypoxic-induced cell invasion includes factors in addition to ADAM17.
Our study suggests that Sp1 transcription factor mediates hypoxia-induced ADAM17 expression and proteolytic activity, and contributes to an increase in invasiveness of brain tumor cells under normoxic and hypoxic conditions. These findings suggest that Sp1 may be a novel target for anti-invasive therapies of brain tumor.