Genomic and Epigenomic Signatures in Ovarian Cancer Associated with Resensitization to Platinum Drugs

Abstract

DNA methylation aberrations have been implicated in acquired resistance to platinum drugs in ovarian cancer. In this study, we elucidated an epigenetic signature associated with platinum drug resensitization that may offer utility in predicting the outcomes of patients who are coadministered a DNA methyltransferase inhibitor. The ovarian cancer specimens we analyzed were derived from a recent clinical trial that compared the responses of patients with recurrent platinum-resistant ovarian cancer who received carboplatin plus the DNA methyltransferase inhibitor guadecitabine or a standard-of-care chemotherapy regimen selected by the treating physician. Tumor biopsies or malignant ascites were collected from patients before treatment (day 1, cycle 1) or after treatment (after 2 cycles) for epigenomic and transcriptomic profiling using the Infinium HumanMethylation450 BeadChip (HM450). We defined 94 gene promoters that were hypomethylated significantly by guadecitabine, with 1,659 genes differentially expressed in pretreatment versus posttreatment tumors. Pathway analysis revealed that the experimental regimen significantly altered immune reactivation and DNA repair pathways. Progression-free survival correlated with baseline expression levels of 1,155 genes involved in 25 networks. In functional investigations in ovarian cancer cells, engineered upregulation of certain signature genes silenced by promoter methylation (DOK2, miR-193a, and others) restored platinum drug sensitivity. Overall, our findings illuminate how inhibiting DNA methylation can sensitize ovarian cancer cells to platinum drugs, in large part by altering gene expression patterns related to DNA repair and immune activation, with implications for improving the personalized care and survival outcomes of ovarian cancer patients.Significance: Epigenomic targeting may improve therapeutic outcomes in platinum-resistant and recurrent ovarian cancer in part by effects on DNA repair and antitumor immune responses. Cancer Res; 78(3); 631-44. ©2017 AACR.

Figure 1. Study population

A) Study design includes phase 1 (cohorts I and II) and randomized phase 2. AUC: area under the curve; G+C: guadecitabine + carboplatin; TC: treatment choice. B) Treatment administration schema and timing of tumor biopsies. C) Summary of sample collection.

Figure 2. Methylome changes induced by guadecitabine

A) Volcano plots of CpG site methylation analysis. Plots of log P value versus log₂ fold change for paired (C1D1 vs. C2D8) biopsies (tumors, left; ascites, right). See Supplemental Table S1 for full list of significantly demethylated genes. B) Pathways enriched by significantly demethylated genes induced by guadecitabine (adjusted P value<0.05) in paired biopsies (tumors, left; ascites, right). C) Two top networks constructed by Immune-Response-Related and DNA Replication and Repair induced by guadecitabine in paired biopsies (tumors, left; ascites, right). D) Pie charts showing the distribution of genomic context of the differentially methylated CpG sites examined (left and right charts show genomic context relative to a nearby CpG island or a gene, which defined by Illumina manifest).

Figure 3. Transcriptomic changes induced by guadecitabine

A) Heatmap shows significant transcriptomic changes induced by guadecitabine. Patient IDs are listed on top of the heatmap, and C1D1 (baseline) is coded in green, while C2D8 (post-treatment) is coded in orange (genes shown here are adjusted P<0.05, |fold change|>2). B) Selected immuno-related pathways enriched by differentially expressed genes (adjusted P value<0.05, |fold change|>2) (full pathways lists are in Supplementary Figure 2 A and B). C) Networks of immune response constructed using significantly changed genes by guadecitabine.

Figure 4. Integrated methylome-transcriptome analysis

A) A heatmap shows 77 integrated genes that were hypomethylated at promoter CpG island and upregulated (mRNA expression) after guadecitabine treatment. C1D1 (baseline) is coded in green, while C2D8 (post-treatment) is coded in orange. Gene list is shown in Supplementary Table S3. B) Immune-related networks constructed by the 77 integrated genes.

Figure 5. Associations between guadecitabine-upregulated genes and clinical outcomes

A) A heatmap shows significant up-regulated genes (associated with PFS) by guadecitabine. Adjusted p value<0.05, fold change>1.3. B) Pathways enriched by 1155 genes whose expression profiles significantly associated with PFS in multivariate Cox regression. C) Pathways enriched by hypomethylated CpG sites in genes associated with PFS. D) Pathways enriched by up-regulated genes that are associated with PFS. Adjusted p value<0.05, fold change>1.3.

Figure 6. Docking protein 2 (DOK2) and microRNA 193a (MIR193A) genes are responsive to DNA demethylation and inhibit proliferation in OC cells

A) Changes in expression of 25 selected genes measured by RNAseq in OC tumors before (n=40) and after (n=9) guadecitabine treatment. The MIR193A and DOK2 genes, which were selected for further validation analyses, are highlighted. B) DOK2 mRNA expression levels measured by real-time RT-PCR in SKOV3, OVCAR3, and OVCAR5 OC cells treated with the hypomethylating agent decitabine (DAC) for 72 hours. C) Real-time RT-PCR measurements of MIR193A mRNA expression levels in SKOV3, OVCAR3, and OVCAR5 OC cells treated with DAC for 72 hours. D) DOK2 mRNA expression levels measured by real-time RT-PCR in SKOV3 cells stably transduced with a vector containing the DOK2 gene. E) Proliferation measured by the CCK8 assay of SKOV3 cells overexpressing or not (Control) the MIR193A gene. F) Survival (CCK8 assay) of SKOV3 cells stably transduced to overexpress the DOK2 gene and treated with cisplatin for 48 hours. G) Clonogenicity of SKOV3 cells overexpressing the MIR193A gene. In B, C, D, E, F and G, experiments were repeated three times, bars represent mean ± SD, n=3. *, P<0.05.

Figure 7. Inhibition of stratifin (SFN gene) in OC cells diminishes cell proliferation and increases sensitivity to platinum

A) Expression levels of the stratifin (SFN) gene in SKOV3 cells stably transfected with control shRNA or shRNA targeting the SFN gene (shSFN, n=3, P<0.05). B) Survival measured by the CCK8 assay of SKOV3 cells transfected with control shRNA or shSFN and treated with cisplatin at the doses indicated (n=3, *P<0.05). C) Clonogenicity of SKOV3 cells transfected with control shRNA or shRNA targeting the SFN gene (shSFN, n=3, *P<0.05). D) Proliferation of SKOV3 cells transfected with control shRNA or shRNA targeting the SFN gene (shSFN, n=3, *P<0.05).