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Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma

Aberrant hedgehog signaling contributes to the development of various malignancies, including glioblastoma (GBM). However, the potential mechanism of hedgehog signaling in GBM migration and invasion has remained to be elucidated. The present study showed that enhanced hedgehog signaling by recombinant human sonic hedgehog N-terminal peptide (rhSHH) promoted the adhesion, invasion and migration of GBM cells, accompanied by increases in mRNA and protein levels of matrix metalloproteinase-2 (MMP-2) and MMP-9. However, inhibition of hedgehog signaling with cyclopamine suppressed the adhesion, invasion and migration of GBM cells, accompanied by decreases in mRNA and protein levels of MMP-2 and -9. Furthermore, it was found that MMP-2- and MMP-9-neutralizing antibodies or GAM6001 reversed the inductive effects of rhSHH on cell migration and invasion. In addition, enhanced hedgehog signaling by rhSHH increased AKT phosphorylation, whereas blockade of hedgehog signaling decreased AKT phosphorylations. Further experiments showed that LY294002, an inhibitor of phosphoinositide-3 kinase (PI3K), decreased rhSHH-induced upregulation of MMP-2 and -9. Finally, the protein expression of glioblastoma-associated oncogene 1 was positively correlated with levels of phosphorylated AKT as well as protein expressions of MMP-2 and -9 in GBM tissue samples. In conclusion, the present study indicated that the hedgehog pathway regulates GBM-cell migration and invasion by increasing MMP-2 and MMP-9 production via the PI3K/AKT pathway.


Introduction
Glioma, the most common type of primary neoplasm of the brain, is the main cause of brain tumor-associated mortality. It can be divided into four sub-types according to the World Health Organization's classification of central nervous system tumors published in 2007 (1). Glioblastoma (GBM), the most malignant type of glioma, accounts for more than half of all gliomas in adults (2). Despite enormous efforts in the development and application of GBM therapies, the prognosis of patients with GBM remains poor due to the highly migratory and invasive nature of GBM cells (3,4). The identification of the molecular mechanisms underlying the aggressive biological behavior of GBM may aid in the development of novel effective therapeutics with the aim of improving the prognosis of patients with GBM.
The hedgehog signaling pathway is essential for embryonic patterning and cancer development (5)(6)(7). The hedgehog protein family includes desert hedgehog, Indian hedgehog and sonic hedgehog (SHH). Among them, SHH is the most widely expressed and the most potent protein. Once SHH binds to its receptor Patched 1, the transmembrane protein Smoothened is released, causing the transport of the transcription factor glioma-associated oncogene (Gli) into the nucleus, where it regulates the transcription of target genes that control cell differentiation, survival and growth (5,6). Three Gli genes (Gli1, Gli2 and Gli3) have been identified in mammalian tissues, among which Gli1 is considered to be the only loyal marker of hedgehog pathway activity (8)(9)(10). Several recent

Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma
studies have shown that the hedgehog pathway is associated with the migration and invasion of ovarian, pancreatic, esophageal and gastric carcinomas (6,(11)(12)(13). However, to date, the implication of the hedgehog pathway in the aggressiveness of GBM has not been elucidated. Furthermore, the mechanisms by which the hedgehog signaling pathway is involved in GBM migration and invasion require further elucidation. Matrix metalloproteinase-2 (MMP-2) and MMP-9, zinc-dependent endopeptidases, are able to degrade nearly all extracellular matrix components to promote cancer-cell invasion (14). It has been demonstrated that MMP-2 and MMP-9 are mostly associated with migration and invasion in multiple tumor types (14)(15)(16). Further studies have shown that the expression of MMP-2 and MMP-9 in several cancer types is mediated by numerous signaling pathways, which include the hedgehog signaling pathway, the wnt/β-catenin pathway, the phosphoinositide-3 kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway (10,17,18). It has been suggested that the hedgehog signaling pathway is cross-linked with other signaling pathways. However, in GBM, it has remained elusive whether there is a link between the hedgehog signaling pathway and the PI3K/AKT pathway or whether the hedgehog signaling pathway regulates the expression of MMP-2 and MMP-9 via the PI3K/AKT.
The present study examined the role of the hedgehog pathway in the migration and invasion of GBM, specifically focusing on the link between the hedgehog signaling pathway and the PI3K/AKT signaling pathway. Specifically, the present study assessed whether the hedgehog pathway is involved in human GBM migration and invasion by induction of MMP-2 and MMP-9 via the PI3K/AKT signaling pathway.

Materials and methods
Human samples. Fifty-one patients with GBM who underwent surgery at the Second Affiliated Hospital of Harbin Medical University (Harbin, China) between March 2010 and July 2013 were enrolled in the present study. All tumor specimens were pathologically diagnosed as GBM. Written informed consent was given by the patients for their information to be stored in the hospital database and used for research. Cell lines and culture conditions. The human glioma U251 cell line, purchased from the Chinese Academy of Science Cell Bank (Shanghai, China), was grown in DMEM/F12 supplemented with 100 U/ml penicillin (Beyotime Institute of Biotechnology, Haimen, China), 100 mg/ml streptomycin (Beyotime Institute of Biotechnology) and 10% fetal bovine serum (FBS; GE Healthcare Life Sciences) in a humidified incubator (5% CO 2 and 37˚C).
Cell adhesion assay. The U251 cells were incubated for 24 h with various concentrations of cyclopamine (0, 5.0 and 10.0 µm) or rhSHH (0, 0.5 and 1.0 µg/ml). The 96-well plates were coated with 5 mg/ml fibronectin (Sigma-Aldrich) and blocked with 1% bovine serum albumin (ZSGB-BIO, Beijing, China) for 4 h. U251 cells (20,000 cells/well) were then seeded into the 96-well plates and incubated for 1 h at 37˚C in 5% CO 2 . Subsequently, the medium was gently removed by aspiration and the wells were washed twice with phosphate-buffered saline (PBS) to remove any non-adherent cells. The adherent cells were quantified using an MTT assay, as we previously described (19).
Migration and invasion assay. The U251 cells were either untreated or pre-treated with GM6001 (15 µm), anti-MMP-2 (20 µg/ml) or anti-MMP-9 neutralizing antibodies (20 µg/ml) for 1 h. The cell invasion assay was performed using a 24-well Transwell chamber (cat no. 3422; Corning-Costar Inc., Corning, NY, USA) with a pore size of 8 µm. Polycarbonate filters were coated with 20 µl diluted Matrigel (1:1 in DMEM/F12; Corning Incorporated, Corning, NY, USA) on the upper side. 1x10 5 cells/well were suspended in 0.2 ml serum-free medium with various concentrations of cyclopamine (0, 5.0 and 10.0 µm) or rhSHH (0, 0.5 and 1.0 µg/ml), and then seeded in the upper chamber, while medium (0.5 ml) containing 10% FBS as a chemoattractant was added to the lower chamber. After a 24-h incubation period at 37˚C with 5% CO 2 , the non-invaded cells and the Matrigel on the upper surface of the filter were removed using a cotton swab and cells on the lower surface of the filter were fixed with methanol (Beyotime Institute of Biotechnology) for 10 min, stained with hematoxylin (ZSGB-BIO, Beijing, China) for 2 min, rinsed in running tap water for 10 min, differentiated with 0.3% acid alcohol (ZSGB-BIO) for 1 min, rinsed in running tap water for 10 min, and finally stained with eosin (ZSGB-BIO) for 2 min. The migration assay was performed as described in the invasion assay but with a shorter incubation period (12 h) and no matrigel coating. A Leica DM2700 M light microscope (Leica Microsystems GmbH, Wetzlar, Germany) was used for evaluating cell number. The cell number on the lower surface of the filter was evaluated by counting three random microscopic fields (magnification, x400) and the numbers of migrated or invaded cells were averaged.

Statistical analysis.
Values are expressed as the mean ± standard deviation. SPSS 19.0 statistical software (International Business Machines, Armonk, NY, USA) was used for statistical analysis. Statistical significance was determined by Student's t-test or Spearman's correlation test. P<0.05 was considered to indicate a statistically significant difference between values.

Inhibition of hedgehog signaling inhibits the adhesion, invasion and migration of GBM cells.
Cyclopamine, a specific antagonist of the hedgehog pathway, was used to evaluate the effects of the hedgehog pathway on the adhesion, invasion and migration of U251 cells. The results showed that cyclopamine suppressed the adhesion of U251 cells in a dose-dependent manner (Fig. 1A). Furthermore, Transwell migration and invasion assays were performed following treatment of the cells with 0, 5.0 or 10.0 µM cyclopamine for 12 h (cell migration) and 24 h (cell invasion), respectively. As demonstrated in Fig. 1B and C, cyclopamine significantly reduced U251-cell migration and invasion in a dose-dependent manner. These results demonstrated that inactivation of the hedgehog pathway markedly inhibited the adhesion, invasion and migration of U251 cells.

Enhancement of hedgehog signaling enhances the adhesion, invasion and migration of GBM cells.
To determine the effects of hedgehog signaling on cell attachment, U251 cells were treated with rhSHH and their adhesion ability was evaluated. As illustrated in Fig. 2A, rhSHH dose-dependently increased the adhesion ability of U251 cells. Furthermore, Transwell migration and invasion assays were performed following treatment of the cells with 0, 0.5 or 1.0 µg/ml rhSHH for 12 h (cell migration) or 24 h (cell invasion), respectively. As shown in Fig. 2B and C, the number of migrated or invaded cells was increased by rhSHH in a dose-dependent manner. These results demonstrated that activation of the hedgehog pathway markedly increased the adhesion, invasion and migration of U251 cells.

Hedgehog signaling enhances cell migration and invasion by increasing the expression of MMP-2 and MMP-9 in GBM.
It is known that MMP-2 and MMP-9 have important roles in tumor migration and invasion (14)(15)(16). Therefore, the present study investigated whether the hedgehog pathway regulated the  expression of MMP-2 and MMP-9. It was observed that treatment with rhSHH dose-dependently upregulated the mRNA levels of MMP-2 and MMP-9 in U251 cells (Fig. 3A), while cyclopamine treatment decreased the mRNA levels of MMP-2 and MMP-9 in U251 cells in a concentration-dependent manner (Fig. 3B). Similarly, rhSHH dose-dependently upregulated the protein levels of MMP-2 and MMP-9 in U251 cells (Fig. 3C), while cyclopamine treatment decreased the protein levels of MMP-2 and MMP-9 in U251 cells in a concentration-dependent manner (Fig. 3D). Furthermore, MMP-2 neutralizing antibody (anti-MMP-2), MMP-9 neutralizing antibody (anti-MMP-9) or GAM6001 were able to inhibit the rhSHH-induced cell migration and invasion ( Fig. 3E and F). These results demonstrated that hedgehog signaling enhanced the expression of MMP-2 and MMP-9 to promote cell migration and invasion of GBM.
Hedgehog signaling regulates the expression of MMP-2 and MMP-9 through the PI3K/AKT pathway in GBM cells. In human glioma cells, the PI3K/Akt pathway is one of the major signaling pathways for the invasive process. Therefore, the present study determined whether activation of the hedgehog pathway is associated with alterations of the PI3K/Akt pathway in GBM. Fig. 4A and B shows that AKT phosphorylation was significantly enhanced by rhSHH, while it was significantly inhibited by cyclopamine. However, the total protein expression of AKT was not altered by rhSHH and cyclopamine. To confirm whether MMP-2 and MMP-9 expression, mediated by the hedgehog pathway, was associated with the PI3K/AKT pathway in GBM cells, this pathway was blocked using PI3K inhibitor Ly294002. Fig. 4C and D shows that Ly294002 significantly decreased rhSHH-induced mRNA and protein expression of MMP-2 and MMP-9 as well as phosphorylation of AKT. These results revealed that hedgehog signaling regulated the expression of MMP-2 and MMP-9 through the PI3K/AKT pathway in GBM cells.

Discussion
Glioblastoma is the most common type of malignant tumor of the central nervous system. Migration and invasion are the leading reasons for treatment failure and cancer-associated mortality of GBM patients (3,4). Therefore, it is necessary to develop novel prevention and treatment strategies for GBM, for which an enhanced knowledge of the underlying molecular mechanisms of migration and invasion of GBM is required. The present study posed and verified the hypothesis that the hedgehog signaling pathway activates the PI3K/AKT pathway, which subsequently upregulates the expression of MMP-2 and MMP-9 at the mRNA and protein level, thereby mediating GBM migration and invasion. The hedgehog signaling pathway has a vital role in the control of cell differentiation, cell proliferation and embryonic development (5,7,12). According to recent studies, aberrant activation of hedgehog signaling is linked to the aggressiveness of multiple tumor types, including GBM (21,22). The results of the present study are therefore consistent with those of previous studies. The present study demonstrated that activation of hedgehog signaling by rhSHH promoted the adhesion, invasion and migration of GBM cells, whereas inhibition of this signaling with cyclopamine, an antagonist of the hedgehog pathway, suppressed the adhesion, invasion and migration of GBM cells. These results suggested that the hedgehog signaling pathway has a tumor-promoting role in GBM that regulates cell invasion and migration.
MMP-2 and MMP-9 are important members of the MMP family, which and promote cancer-cell migration and invasion by degrading extracellular matrix components (2,(14)(15)(16)(17). An increasing number of studies demonstrated that MMP-2 and MMP-9 have important roles in GBM angiogenesis and can be regulated by multiple pathways (23)(24)(25). The present study examined MMP-2 and MMP-9 expression to explore the association between the hedgehog pathway and cell invasion and migration. It was found that rhSHH dose-dependently upregulated mRNA and protein levels of MMP-2 and MMP-9 in U251 cells, whereas cyclopamine decreased mRNA and protein levels of MMP-2 and MMP-9 in U251 cells in a concentration-dependent manner. In addition, U251 cells were treated with MMP-2-or MMP-9-neutralizing antibody or with GAM6001, and their effects on rhSHH-induced migration and invasion of GBM cells was evaluated. A Transwell assay revealed that MMP-2-and MMP-9 neutralizing antibody or GAM6001 were able to reverse rhSHH-induced cell migration and invasion. These results indicated that hedgehog signaling Table I. Correlation between Gli1, phospho-AKT, MMP-2 and MMP-9 protein expression.  promotes cell migration and invasion by enhancing the expressions of MMP-2 and MMP-9 in GBM cells. Accumulating evidence revealed that the PI3K/Akt pathway is activated in multiple types of cancer, including kidney, lung, liver and ovarian cancer, and has an important role in migration and invasion (26)(27)(28). Several studies reported that the PI3K/Akt pathway is mediated by the SHH pathway. For example, Yoo et al (29) reported that hedgehog signaling promotes the metastasis of gastric cancer through activation of the PI3K/Akt pathway. Morton et al (30) also demonstrated that inhibition of the hedgehog pathway markedly regulated pancreatic tumorigenesis through inhibition of the PI3K/AKT pathway. However, it remains elusive whether such association also exists in GBM. In the present study, AKT phosphorylation of GBM cells was significantly enhanced by rhSHH and was significantly inhibited by cyclopamine, verifying the hypothesis that the PI3K/Akt pathway is mediated via the hedgehog pathway in GBM cells. Furthermore, the present study demonstrated that the PI3K inhibitor Ly294002 significantly decreased rhSHH-induced MMP-2 and MMP-9 mRNA and protein expression as well as phosphorylation of AKT. More importantly, immunohistochemical analysis showed that Gli1 protein expression was closely correlated with phospho-AKT, MMP-2 and MMP-9 in GBM samples. All of these results suggested that hedgehog signaling regulates the expression of MMP-2 and MMP-9 through the PI3K/AKT pathway in GBM.
In conclusion, the present study demonstrated for the first time, to the best of our knowledge, that the hedgehog signaling pathway promotes the invasion and migration of GBM cells by enhancing MMP-2 and MMP-9 expression via the PI3K/AKT pathway. These results provided a novel molecular mechanism of GBM-cell aggressiveness based on the hedgehog signaling pathway and may assist in the discovery of novel therapeutic targets to control the aggressiveness of GBM.