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, 4 (2), e4563

Integrative Analysis of Epigenetic Modulation in Melanoma Cell Response to Decitabine: Clinical Implications

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Integrative Analysis of Epigenetic Modulation in Melanoma Cell Response to Decitabine: Clinical Implications

Ruth Halaban et al. PLoS One.

Abstract

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21(Cip1) in a p53-independent manner, identified the TGFbeta pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated beta-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient's selection and monitoring response, as well as targets for improved combination therapy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cellular responses to Aza.
Panel A. Growth arrest in response to Aza. Melanoma cells were untreated or treated with increasing concentrations of Aza for 2 days (under line), released into regular growth medium and counted at 2–3 days intervals. The figure shows representative growth curves of a sensitive (YUMAC) and resistant (501 mel) melanoma cell strains of two biological replicates. Supplemental data provide the growth curves (Figure S1) and the population doubling time (Table S1) of all cell strains. Panel B. Aza IC50 response. The vertical line separates the designated sensitive (top) and resistant cell cells (bottom). Panel C. Apoptosis in response to low-dose Aza (0.2 µM) measured by the Caspase-Glo 3/7 assay kit. Panel D. Apoptosis in response Aza (0.2 µM) detected by immunofluorescence with anti-caspase-3 active rabbit antibodies (green arrows point at green fluorescing apoptotic cells). The cell nuclei are stained with DAPI (blue). Bars indicate 20 µm. The histogram shows percent apoptotic cells measured by counting the number of active caspase-3 positive green fluorescing cells in 10 independent microscopic fields representing about 800 cells each. The cell base assay shows a lower percentage of apoptotic cells in response to Aza compared to Panel C because large numbers of affected cells detached during the staining and washing procedures.
Figure 2
Figure 2. Bioinformatic analysis of whole genome expression arrays.
Panel A. Unsupervised hierarchical clustering of absolute intensity values. The vertical scale indicates 1-pearson's correlation coefficients as a measure of similarity. Panel B. Heatmap of differentially expressed sequences after treatment with low-dose Aza. Panel C. DNMT1 expression at the end of 3-days treatment with Aza (0.2 µM). Cell extracts were subjected to Western blot with anti-DNMT1 antibodies. The same membrane was successively blotted with anti-b-actin antibodies as a measure for protein load in each well. Panel D. Pie chart of the most over-represented Gene Ontology terms (p-value<1e-3); the size is relative to the number of represented genes, and the color represents the enrichment p-value. Panel E. SFRP1 transcripts in melanoma cell strains as assessed by the oligonucleotide array hybridization. The data represent one of two sequence IDs with similar results. The error bars represent the Standard Deviations (SD). One, two, three stars refer to p-value less than 0.05, 0.01 or 0.001, respectively. We determined p-values by unpaired t-test (Aza vs. Untreated). The broken line in this and all subsequent figures separates sensitive (left hand side) from resistant (right hand side) cell strains.
Figure 3
Figure 3. Activated Wnt/β-catenin/MITF pathway confers resistance to Aza.
Panel A. Chromatograms showing CTNNB1 activating mutations in 501 mel cells (GAC/CAC) and YURIF tumor (TCT/TGT) (marked by brackets and arrows), which lead to D32H and S33C mutations, respectively. The same results were obtained with YURIF short term cultured cells. Panel B. Expression of β-catenin and MITF in melanoma cell strains relative to β-actin. CTNNB1 mutation status for each cell strain is indicated at the top. Panel C. siRNA knockdown of β-catenin and downstream targets. Parallel cultures were untreated or treated with Aza (0.2 µM) for 2 days followed by transient transfection with three different CTNNB1 siRNA or with Alexa Fluor as a control for one day. Cell extracts were subjected to successive Western blotting with β-catenin, MITF, BCL2, Myc, and β-actin. Panel D. The same cultures as in panel C were assessed for apoptosis employing the Caspase 3/7 assay. Bars indicate SD of 3 replicate wells.
Figure 4
Figure 4. TP53-independet CDKN1A reactivation and promoter methylation.
Panel A. Reactivation of CDKN1A in melanoma cells in response to Aza (0.2 µM) as revealed by oligonucleotide array hybridization. The data represent one of two sequence IDs with similar results. All other details as in Figure 2 Panel E. Panel B. Expression of p21Cip1 and p53, with β-actin serving as a control. Parallel cultures of melanoma cells were untreated (−) or treated (+) with Aza (0.2 µM). MG132 (20 µM) was added 6 h prior to harvesting the cells where indicated (+). The levels of p53 protein were in agreement with gene transcript levels showing that TP53 was inactivated in YUMAC (absolute hybridization intensities values of ∼220, compared to 8,000–12,000 in the melanoma cell strains). Here and in all other Western blots numbers on the left mark the location of prestained protein markers in KDa, heavy and light frames designate Aza resistant and sensitive cells, respectively. Panel C. BS sequencing results of CDKN1A proximal promoter (−214 to +20 relative to TSS). Melanoma cells were untreated (control), and Aza (0.2 µM) treated as described in Panel B. Symbols: Arrows indicate the TSS; open and black circles, unmethylated and methylated CG pairs, respectively; dark and light grey circles indicate about 50% and 10% methylated CG, respectively. Numbers on the bottom indicate bp location relative to TSS.
Figure 5
Figure 5. CLU reactivation and promoter methylation.
Panel A. CLU re-expression in melanoma cells in response to Aza (0.2 µM) as assessed by the oligonucleotide array hybridization. The data represent one of three sequence IDs with similar results. All other details as in Figure 2 Panel E. Panel B. Clusterin expression as revealed by Western blots with anti-CLU antibodies. The results are representative of two biological replicas. Panel C. BS DNA sequencing results of the proximal CLU promoter and part of first exon regions. The bar indicated the CG island and the arrows the site of primers used for amplification. All other details as in Figure 4.
Figure 6
Figure 6. Reactivation of CDKNA1 and CLU by histone acetylation.
The Western blots show p21Cip1 (Panel A) and CLU (Panel B) expression in YUMAC melanoma cells treated with increasing concentrations of Trichostatin A (TSA) overnight, as revealed by probing with the respective antibodies using β-actin as a control (left panel). Middle panels show expression of p21Cip1 and CLU after 2-days treatment with Aza (0.2 µM), where indicated (+) followed by 1-day incubation with PXD101 (1 µm). Left panels: Real-Time PCR data comparing fold-difference in CDKN1A and CLU transcript in YUMAC and 501 mel cells after treatment with low-dose Aza and PXD101, alone and in combination, compared to non-treated cells. PXD101 (1 µM) was added for 24 hrs before harvesting the cells. Notice differences in scale of absolute hybridization intensities in YUMAC and 501 mel cells. However, 501 mel expressed about 370 fold less CDKN1A gene transcripts compared to YUMAC cells, apparently insufficient to lead to detectable p21Cip1 protein. The basal levels of CLU transcripts in YUMAC were about 50 fold higher relative to 501 mel melanoma cells, in agreement with low protein levels (data not shown). Panel C. Growth response to combination treatment with HDAC inhibitors Trichostatin A (TSA) and PXD101. The sensitive YUMAC and resistant 501 mel cells were incubated in triplicate wells without or with Aza (0.2 µM and 0.4 µM as indicated) followed by one-day recovery in regular medium, or medium supplemented with increasing concentrations of TSA (a), or PXD101 (b). Cell viability was assessed with the CellTiter-Glo® Luminescent Cell Viability Assay. Data are presented as percent of control, non-treated cells.
Figure 7
Figure 7. TGFBI reactivation by promoter demethylation.
Panel A. TGFBI re-expression in response to Aza (0.2 µM) as assessed by the oligonucleotide array hybridization. The heavy line on the ordinate represent the levels of TGFBI- transcript levels in adult melanocytes. The data represent one sequence ID. All other details as in Figure 2 panel E. Panel B. Validation of TGFBI re-expression at the protein level by Western blots with anti-TGFBI antibodies employing b-actin as a control. The results are representative of two biological replicas. Panel C. BS sequencing results of TGFBI proximal promoter and first exon in normal human melanocytes (NBMel) and melanoma cells untreated (control), and Aza (0.2 µM) treated cells. Panel D. Chromatograms of the distal promoter about -50 to −100 bp downstream of TSS as shown in C. Boxed nucleotide pairs indicate position of intact (CG), partially BS modified (C/TG) and deaminated (TG) CG pairs. All other details as in Figure 4.
Figure 8
Figure 8. Validation of CLU and TGFBI apoptotic activity.
Panel A. Reduction of Clu and TGBFI proteins by gene specific siRNA knockdown assessed by Western blotting. YUMAC melanoma cells were untreated or treated with Aza (0.2 µM) for 2 days followed by transient transfection with siRNA directed to Clu, TGFBI, or a mixture of the two, employing Alexa Fluor as a control, as indicated. Cells were harvested the following day and extracts subjected to successive Western blotting with the respective antibodies, and anti-β-actin as a control. Panel B. Parallel cultures were tested for apoptosis with the Caspase 3/7 assay. Values are given as percent of control, i.e., non-transfected cultures.
Figure 9
Figure 9. Bortezomib Augments the Aza growth-arrest response in a synergistic manner.
Panel A. Dose-dependent effects of Bortezomib on cell proliferation. YUMAC melanoma cells were seeded in 24 well plates, incubated with increasing concentration of Bortezomib for 72 h, and cells from triplicate wells were harvested and counted with Coulter counter. Panels B and C. Growth responses of 501 mel and YURIF melanoma cells to 0.2 µM Aza (red), 2 nM Bortezomib (green) and combination (purple), compared to non-treated cells (black). Values are average of duplicate wells. Bars indicate double standard errors that ranged between 5–10% of total counts. Similar results were obtained with 1 nM Bortezomib in combination with Aza. The figures show one of two replicate experiments with similar results.

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