GADD45gamma: a new vitamin D-regulated gene that is antiproliferative in prostate cancer cells

Abstract

1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of normal and malignant prostate epithelial cells at least in part through inhibition of G1 to S phase cell cycle progression. The mechanisms of the antiproliferative effects of 1,25-(OH)2D3 have yet to be fully elucidated but are known to require the vitamin D receptor. We previously developed a 1,25-(OH)2D3-resistant derivative of the human prostate cancer cell line, LNCaP, which retains active vitamin D receptors but is not growth inhibited by 1,25-(OH)2D3. Gene expression profiling revealed two novel 1,25-(OH)2D3-inducible genes, growth arrest and DNA damage-inducible gene gamma (GADD45γ) and mitogen induced gene 6 (MIG6), in LNCaP but not in 1,25-(OH)2D3-resistant cells. GADD45γ up-regulation was associated with growth inhibition by 1,25-(OH)2D3 in human prostate cancer cells. Ectopic expression of GADD45γ in either LNCaP or ALVA31 cells resulted in G1 accumulation and inhibition of proliferation equal to or greater than that caused by 1,25-(OH)2D3 treatment. In contrast, ectopic expression of MIG6 had only minimal effects on cell cycle distribution and proliferation. Whereas GADD45γ has been shown to be induced by androgens in prostate cancer cells, up-regulation of GADD45γ by 1,25-(OH)2D3 was not dependent on androgen receptor signaling, further refuting a requirement for androgens/androgen receptor in vitamin D-mediated growth inhibition. These data introduce two novel 1,25-(OH)2D3-regulated genes and establish GADD45γ as a growth-inhibitory protein in prostate cancer. Furthermore, the induction of GADD45γ gene expression by 1,25-(OH)2D3 may mark therapeutic response in prostate cancer.

Figure 1

1,25-(OH)₂D₃-mediated induction ofGADD45γ and MIG6 is associated with growth inhibition in LNCaP cells. A, Parental LNCaP and derivatives VitD.R and Con.R cells were treated with either 50 nm 1,25-(OH)₂D₃ or EtOH vehicle for 72 h. Cell numbers were determined and the percentage inhibition of cell proliferation was calculated. Mean percent inhibition (±sem) of three experiments performed in triplicate is shown. B, Venn diagrams of microarray results. C and D, Reverse transcriptase real-time PCR using Taqman probes for GADD45γ (C) and MIG6 (D) and normalized to 18S (control) was performed 24 h after 50 nm 1,25-(OH)₂D₃ or vehicle treatment. The mean induction of a total of three experiments performed in triplicate (±sem) is shown.

Figure 2

Ectopic expression of GADD45γ but not MIG6 causes growth inhibition and G₁ accumulation of LNCaP cells. A and B, Proliferation and cell cycle distribution of LNCaP cells stably expressing GADD45γ or MIG6 was compared with that of GFP-expressing cells (as a control) and with LNCaP cells treated for 72 h with 50 nm 1,25-(OH)₂D₃ vs. EtOH vehicle. The mean percent growth inhibition (±sem) of a total of three experiments performed in triplicate is shown in A. The results of flow cytometry for a representative experiment (of three) performed in triplicate are shown in B as a mean percentage of the cells in each phase of the cell cycle (±sem). C, LNCaP cells expressing GFP, GADD45γ, and MIG6 and untransfected LNCaP cells treated with 50 nm 1,25-(OH)₂D₃ or ETOH vehicle were harvested 72 h after infection with lentiviral constructs. Rb (pRb), hyperphosphorylated Rb (ppRb), cyclin A, and actin were detected by Western blotting. The Western blots shown are representative of a total of three experiments.

Figure 3

GADD45γ was not induced by 1,25-(OH)₂D₃ in resistant ALVA31 cells. A, Proliferation of ALVA31 cells treated with either 50 nm 1,25-(OH)₂D₃ or EtOH vehicle for 72 h was evaluated. The data are representative of a total of three experiments done in triplicate (±sd). B, Reverse transcriptase real-time PCR using Taqman probes for GADD45γ and MIG6 and normalized to 18S (control) was performed 24 h after 50 nm 1,25-(OH)₂D₃ treatment. The mean fold induction (±sem) of a total of three experiments performed in triplicate is shown. C, A representative experiment (done in triplicate) of proliferation of ALVA31 cells stably expressing GADD45γ, MIG6, or GFP. D, ALVA31 cells stably expressing GADD45γ, MIG6, or GFP and untransfected ALVA31 cells treated with EtOH vehicle or 50 nm 1,25-(OH)₂D₃ for 72 h were analyzed by flow cytometry. Mean percentage of cells in each phase of the cell cycle (±sem) is shown for a representative experiment (performed in triplicate) of three total experiments. All statistical analyses were done using the Student’s t-test, *, P < 0.05; **, P < 0.01.

Figure 4

AR signaling is not involved in the up-regulation of GADD45γ by 1,25-(OH)₂D₃. A, Reverse transcriptase real-time PCR using Taqman probes for GADD45γ and PSA and normalized to 18S (control) was performed 24 h after 50 nm 1,25-(OH)₂D₃, 10 μm bicalutamide, or EtOH vehicle treatment. The results of a total of three experiments performed in triplicate are shown (±sem) (comparisons are to vehicle-treated controls). B, LNCaP cells infected with lentiviruses encoding either short hairpin AR or a scrambled control were harvested 72 h after infection and AR and actin were detected by Western blotting. The Western blot shown is representative of a total of three experiments. C, Reverse transcriptase real-time PCR using Taqman probes for GADD45γ and PSA normalized to 18S (control) was performed 24 h after 50 nm 1,25-(OH)₂D₃ treatment. The results of a total of three experiments performed in triplicate are shown (±sem) [comparisons are to vehicle-treated LNCaP cells (*, P < 0.05; **, P < 0.01)].

Figure 5

GADD45γ induction occurs in as little as 3 h yet requires de novo protein synthesis. A, Reverse transcriptase real-time PCR using Taqman probes for GADD45γ and normalized to 18S (control) was performed 24 h after 50 nm 1,25-(OH)₂D₃ or vehicle treatment of LNCaP cells. The results of a total of three experiments performed in triplicate is shown experiment (±sem). B, LNCaP cells were treated with 10 μg/ml cycloheximide for 30 min and then treated with 50 nm 1,25-(OH)₂D₃ for 24 h. Reverse transcriptase real-time PCR using Taqman probes for GADD45γ and normalized to 18S (control) was performed. The results of a total of three experiments performed in triplicate are shown (±sem) [comparisons are to vehicle-treated controls (*, P < 0.05; **, P < 0.01)].

Figure 6

GADD45γ induction by 1,25-(OH)₂D₃ predicts sensitivity to 1,25-(OH)₂D₃-mediated growth inhibition. A–C, Reverse transcriptase real-time PCR using Taqman probes for GADD45γ (A), MIG6 (B), and CYP24A1 (C) and normalized to 18S (control) was performed 24 h after 50 nm 1,25-(OH)₂D₃ or vehicle treatment of the indicated cell lines. The results of a total of three experiments performed in triplicate are shown experiment (±sem).