Role of GLI2 transcription factor in growth and tumorigenicity of prostate cells

Abstract

Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including prostate cancer. The GLI2 transcription factor is a primary mediator of Hh signaling. However, its relative contribution to development of prostate tumors is poorly understood. To establish the role of GLI2 in maintaining the tumorigenic properties of prostate cancer cells, we developed GLI2-specific small hairpin RNA. Knockdown of GLI2 in these cells resulted in significant down-regulation of the Hh signaling pathway, followed by inhibition of colony formation, anchorage-independent growth, and growth of xenografts in vivo. Conversely, ectopic expression of Gli2 in nontumorigenic prostate epithelial cells resulted in accelerated cell cycle progression, especially transition through G(2)-M, and augmented proliferation. Altogether, our findings suggest that GLI2 plays a critical role in the malignant phenotype of prostate cancer cells, and GLI2 may potentially become an attractive therapeutic target for the treatment of prostate cancer.

Figure 1. shRNA-mediated inhibition of GLI2 inhibits GLI-dependent transcription in prostate cancer cells

A, immunoblot analysis of GLI2 protein isolated from 293T cells transfected with shRNA plasmids as indicated. Representative of two independent experiments. Prostate cancer cells were transfected with pGL3-Bcl2promo luciferase (B), K17-luciferase (C), 8×3′Gli BS-LucII (D), pSV40 β-gal (Promega), and shRNA expression plasmids as indicated. Luciferase activity was estimated using luciferase reporter assay reagent (Promega). β-Galactosidase was used for normalization and estimated using β-galactosidase assay reagent (Pierce). *, P < 0.01, compared with cells transfected with scrambled shRNA (Student’s t test).

Figure 2. Knockdown of GLI2 suppresses proliferation and anchorage-independent growth of prostate cancer cells

A, the colony formation assay was carried out in RWPE1 and prostate cancer cell lines transfected with scrambled and GLI2 shRNAs. The transfected cells were selected with puromycin (Sigma), allowed to form colonies, fixed with 10% formalin, stained with 2% Gentian violet (Ricca Chemical Company), and analyzed statistically. Anchorage-independent growth of 22Rν1 cells was studied. Cells infected with GLI2 shRNA– or scrambled shRNA–encoding lentiviruses were grown in six-well plates at a concentration of 10,000 per well as described. Colony formation was determined under low magnification (×10) in an inverted microscope (B) and counted (C). Inset, levels of GLI2 expression analyzed by immunoblotting with Gli2 antibody (G20). *, P < 0.01, compared with cells infected with scrambled shRNA (Student’s t test).

Figure 3. GLI2 down-regulation inhibits growth of 22Rν1 xenografts

Tumor growth studies were done in nude mice injected with 22Rν1 cells expressing GLI2 shRNA or scrambled shRNA. The time to a target mean tumor volume of 500 mm³ is defined as the elapsed time from the date of treatment to the date when the 500 mm³ target is reached or when the mouse is sacrificed. A, a linear regression analysis was used to measure both the rate of mean tumor or carcinoma area growth and tumor multiplicity as a function of time using S-plus Software. B, a Kaplan-Meier survival analysis with the corresponding log-rank analysis of Kaplan-Meier data (C) was done using S-plus Software (Insightful), * P < 0.05.

Figure 4. Overexpression of Gli2 accelerates growth of RWPE1 cells

A, cell proliferation assay was measured in RWPE1 cells stably expressing pcDNA3.1 or pcDNA3.1-Flag-Gli2 as indicated. The proliferation was estimated with CellTiter 96 AQueous One Solution Reagent (Promega) 24, 48, and 72 h after the initial reading at 490 nm using an ELISA reader. The levels of GLI2 in these RWPE1 clones were analyzed by immunoblotting with Gli2 antibody (G20, Santa Cruz biotechnology). B, cell cycle analysis was carried in stable RWPE1 clones of pcDNA3.1+ (control) and cells expressing Gli2 by propidium iodide staining. Cells were synchronized by starving on serum-free medium without growth factors and medium supplements for 24 h and then harvested 24 h after addition of serum. Cells fixed with 95% ethanol were treated with RNase A (100 µg/mL) for 30 min at 37°C and with propidium iodide (25 µg/mL; Molecular Probes) for ≥30 min at 37°C for flow analysis.