The hedgehog pathway transcription factor GLI1 promotes malignant behavior of cancer cells by up-regulating osteopontin - PubMed (original) (raw)

The Hh pathway transcriptionally regulates OPN. A, levels of GLI1 and OPN are increased in primary cutaneous cancer and metastatic melanoma. Gene microarray analysis (utilizing a Human Genome U133 Plus 2.0 array from Affymetrix, Inc.) was used to compare 40 metastatic melanoma samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 16 primary cutaneous melanoma specimens (14). The expression levels of GLI1 and OPN increase progressively beyond the stage of MIS through the stage of metastatic melanoma. Thin, thin melanomas (<1.5 mm in Breslow thickness); _IM_, intermediate thickness (between 1.5 and 4.0 mm in Breslow thickness); _thick_, melanomas (that are >4.0 mm in Breslow thickness). The left y axis denotes the scale for GLI1 expression and the right y axis corresponds to OPN levels. As compared with MIS, the increase in GLI1 in the metastatic melanoma samples is statistically significant (p = 0.020). The increase in OPN expression in the thick and metastatic melanoma specimens is statistically significant compared with the corresponding OPN levels in the MIS specimens (p = 0.018 and 0.0018, respectively). B and C, inhibition of the Hh pathway negatively impacts OPN expression in MCC012A, MCC012F, and MDA-MB-435 cells. B, cyclopamine treatment significantly (* indicates p < 0.0001) decreases the levels of OPN mRNA (assessed by qRT-PCR) in MCC012A and MCC012F cells. C, cyclopamine significantly (* indicates p < 0.0001) decreases the levels of OPN mRNA in a dose-dependent manner in MDA-MB-435 cells. Specifically, cells were treated with the indicated concentrations of cyclopamine in Dulbecco's modified minimum essential medium, F-12 supplemented with 0.5% fetal bovine serum. This medium was replaced with fresh cyclopamine-containing medium after 12 h. Cells were harvested for assay after 24 h of cyclopamine treatment. RNA was assessed by real-time RT-PCR for OPN transcript levels. D, cyclopamine causes a dose-dependent decrease in the OPN promoter activity (200 ng of pGL3-OPN transfected) in MCC012A and MCC012F cell lines. In the MCC012F cells, at the doses tested (10 and 20 μ

m

), cyclopamine caused a significant (p = 0.042 and 0.002, respectively) decrease in OPN promoter activity. In the MCC012A cell line, cyclopamine (10 μ

m

) caused a noticeable, but not significant (p = 0.06) decrease in OPN promoter activity. Treatment with 20 μ

m

cyclopamine caused a significant decrease (p = 0.0006) in OPN promoter activity. Tomatidine had no effect on the promoter activity of OPN. E, levels of OPN in the secretome were decreased upon treatment with cyclopamine. MDA-MB-435 cells were grown for the indicated time in 0 (dimethyl sulfoxide (DMSO) control), 10, and 20 μ

m

cyclopamine. The conditioned media were assayed for OPN. F and G, the Hh ligands stimulate OPN promoter activity. Cells (MDA-MB-435) were transfected with the OPN promoter construct (200 ng) and treated with increasing concentrations of either SHH (F) or IHH (G). The asterisk above the graph indicates that the activity of the OPN promoter was significantly (p < 0.0001) higher than that of the corresponding control (untreated) group for all concentrations of IHH and SHH tested. H, triggering the Hh pathway by treatment with the ligands, SHH and IHH, results in a significant increase in OPN promoter activity in metastatic melanoma cell lines, MCC012A (p = 0.0004 for SHH and p < 0.0001 for IHH treatments) and MCC012F (p = 0.0078 for SHH and p = 0.0032 for IHH). I, SHH was able to rescue the inhibitory effects of cyclopamine on OPN transcript levels. MDA-MB-435 cells (1 million) were treated with cyclopamine or tomatidine (20 μ

m

). After 12 h, the medium of one cyclopamine-treated set was replaced with medium containing recombinant SHH (100 n

m

). The experiment was terminated after 24 h of the start of the initial cyclopamine treatment. RNA was assessed by real-time RT-PCR for OPN transcript levels. Error bars represent mean ± S.E.