Epigenetic regulation by decitabine of melanoma differentiation in vitro and in vivo

Abstract

Apoptosis genes, such as TP53 and p16/CDKN2A, that mediate responses to cytotoxic chemotherapy, are frequently nonfunctional in melanoma. Differentiation may be an alternative to apoptosis for inducing melanoma cell cycle exit. Epigenetic mechanisms regulate differentiation, and DNA methylation alterations are associated with the abnormal differentiation of melanoma cells. The effects of the deoxycytidine analogue decitabine (5-aza-2'-deoxycytidine), which depletes DNA methyl transferase 1 (DNMT1), on melanoma differentiation were examined. Treatment of human and murine melanoma cells in vitro with concentrations of decitabine that did not cause apoptosis inhibited proliferation accompanied by cellular differentiation. A decrease in promoter methylation, and increase in expression of the melanocyte late-differentiation driver SOX9, was followed by increases in cyclin-dependent kinase inhibitors (CDKN) p27/CDKN1B and p21/CDKN1A that mediate cell cycle exit with differentiation. Effects were independent of the TP53, p16/CDKN2A and also the BRAF status of the melanoma cells. Resistance, when observed, was pharmacologic, characterized by diminished ability of decitabine to deplete DNMT1. Treatment of murine melanoma models in vivo with intermittent, low-dose decitabine, administered sub-cutaneously to limit high peak drug levels that cause cytotoxicity and increase exposure time for DNMT1 depletion, and with tetrahydrouridine to decrease decitabine metabolism and further increase exposure time, inhibited tumor growth and increased molecular and tumor stromal factors implicated in melanocyte differentiation. Modification of decitabine dose, schedule and formulation for differentiation rather than cytotoxic objectives inhibits the growth of melanoma cells in vitro and in vivo.

Figure 1. Effects on proliferation and morphology

(A) Decitabine 0.5 µM decreased melanoma cell-line but not normal melanocyte proliferation. Human and mouse melanoma cell lines normal human melanocytes (NHM) were cultured with decitabine 0.5 µM added on days 1 and 4. Cell counts by automated counter. Proliferation is expressed relative to vehicle treated control. (B) Decitabine 0.5 µM induced morphologic changes of differentiation. Human and mouse melanoma cell lines and normal human melanocytes (NHM) were cultured in vitro with decitabine 0.5 µM added on days 1 and 4. Images were obtained on day 8 by phase- contrast (left columns of the panel) or Giemsa staining of cytospins of equal number of cells (right columns of the panel). All images identical 100× magnification.

Figure 2. Effects on apoptosis and differentiation pathways

(A) The concentration of decitabine (DAC) used did not induce apoptosis as measured by Annexin/PI-staining 24 hours after addition of drug. Camptothecin (CAM) was used as a positive control for apoptosis. (B) Response of A375 human melanoma cells. A375 cells were cultured with decitabine 0.5 µM added at 0h. Protein levels measured by Western blot. (C) Response of B16 murine melanoma cells. B16 cells were cultured with decitabine 0.5 µM added at 0h. Protein levels measured by Western blot.

Figure 3. Decitabine effects on SOX9 proximal promoter and LINE-1 transposon CpG methylation

(A) SOX9 promoter and LINE-1 repetitive element CpG methylation in normal human melanocytes (NHM). Methylation measured by pyrosequencing. SOX9 promoter CpG are in a conserved region of the proximal promoter (figure S3). (B) SOX9 promoter and LINE-1 CpG methylation in p53 and p16-null MeWo melanoma cells before day 0 and after day 7 decitabine. Decitabine 0.5 µM added on day 1.

Figure 4. Effects on gene expression

Gene expression measured by QRT-PCR on day 0, 4 and 8. Human melanoma cells lines and normal human melanocytes (NHM) were cultured in vitro with decitabine at 0.5 µM on day 1, 4, and 7. (A) MITF and DCT expression. (B) The ratio of DCT/MITF expression. (C) p21/CDKN1A, S100A, TGFB – genes previously reported to be upregulated by decitabine treatment of melanoma cells. (D) S100B, BCL2 – genes previously reported as not being upregulated by decitabine treatment of melanoma cells.

Figure 5. Gene and DNMT1 expression of resistant cells

B16 melanoma cells were maintained in culture with decitabine for 90 days. Decitabine-resistant and wild-type control B16 melanoma cells were analyzed for (A) gene expression by QRT-PCR with and without further exposure to decitabine at 0.5 µM and (B and C) DNMT levels using immunofluorescence with Q-Dots. WT-Unt = wild-type B16 untreated control, WT-DAC = decitabine treated, Res-Unt = decitabine resistant B16 untreated, Res-DAC = decitabine treated.

Figure 6. Effects of decitabine (DAC) and THU (THU) on tumor growth and gene expression in vivo

(A) B16 melanoma cells were implanted s.c. on day 1. Beginning on day 3 groups of mice were treated with THU 30–60 minutes prior to DAC either i.p. or s.c. A group was not treated (NT). Data represent mean tumor volume ± SEM, n = 7 mice per group. (B) A375 cells were implanted s.c. on day 1. Beginning on day 3, mice were treated with THU-DAC s.c. as above. Data represent mean tumor volume ± SEM, n = 7 mice per group. (C) Effects of THU-DAC on intratumoral gene expression in vivo. B16 and A375 cells were implanted s.c. on day 1. Beginning on day 10, mice were treated with decitabine at 0.2 mg/kg 3×/week and THU at 4 mg/kg 2×/week. A group of mice was not treated (NT). Tumor was harvested on day 24. Regulators of MITF and melanocyte differentiation markers were assessed by QRT-PCR (murine and human specific primers were used for B16 and A375 respectively). Values are presented as the ratio of gene expression in treated versus non-treated groups (mean ± SD, n = 4 mice per group).