Yin yang 1 modulates taxane response in epithelial ovarian cancer

Abstract

Survival of ovarian cancer patients is largely dictated by their response to chemotherapy, which depends on underlying molecular features of the malignancy. We previously identified YIN YANG 1 (YY1) as a gene whose expression is positively correlated with ovarian cancer survival. Herein, we investigated the mechanistic basis of this association. Epigenetic and genetic characteristics of YY1 in serous epithelial ovarian cancer were analyzed along with YY1 mRNA and protein. Patterns of gene expression in primary serous epithelial ovarian cancer and in the NCI60 database were investigated using computational methods. YY1 function and modulation of chemotherapeutic response in vitro was studied using small interfering RNA knockdown. Microarray analysis showed strong positive correlation between expression of YY1 and genes with YY1 and transcription factor E2F binding motifs in ovarian cancer and in the NCI60 cancer cell lines. Clustering of microarray data for these genes revealed that high YY1/E2F3 activity positively correlates with survival of patients treated with the microtubule-stabilizing drug paclitaxel. Increased sensitivity to taxanes, but not to DNA cross-linking platinum agents, was also characteristic of NCI60 cancer cell lines with a high YY1/E2F signature. YY1 knockdown in ovarian cancer cell lines results in inhibition of anchorage-independent growth, motility, and proliferation but also increases resistance to taxanes, with no effect on cisplatin sensitivity. These results, together with the prior demonstration of augmentation of microtubule-related genes by E2F3, suggest that enhanced taxane sensitivity in tumors with high YY1/E2F activity may be mediated by modulation of putative target genes with microtubule function.

Figure 1. YY1 target genes in ovarian cancer

(A) Genes whose expression is correlated with that of YY1 are enriched in binding motifs for YY1. Color bar represents all Affymetrix U133A probes with a RefSeq annotation arranged by their correlation to the expression of YY1 using the expression values from 88 ovarian tumors. Black and white bar represents the same distribution of probes, where a single black horizontal line indicates the presence of an YY1 binding motif (V$YY1_Q6) for that gene. (B) Among 4,749 genes that contain the V$YY1_Q6 binding motif, genes whose expression is correlated with that of YY1 are enriched for E2F binding motifs (V$E2F1_Q3). (C) Clustering by putative YY1 target genes in ovarian tumors. The top 250 YY1-positively correlated genes were selected for clustering based on 1) the presence of both YY1 (V$YY1_Q6) and E2F (V$E2F1_Q3) binding motifs and 2) having at least one tumor with relatively high level expression of the gene (RMA normalized value >7). The ‘YY1 Low’ and ‘YY1 High’ clusters are characterized by low and high expression, respectively of YY1 and YY1- correlated genes. Color bar below the heat map, probability of having an ‘E2F3 signature’. (D) Kaplan-Meier analysis of primary ovarian cancer patients who received platinum and did (left; N=36) or did not (right; N=28) receive paclitaxel. Patients are stratified based on the assignment of their tumor to the ‘YY1 Low’ or ‘YY1 High’ cluster.

Figure 2. YY1 target genes in the NCI60 cell lines

(A) Genes whose expression is correlated with that of YY1 are enriched in binding sites for YY1. Color bar represents all U95Av2 probes with a RefSeq annotation arranged by their correlation to the expression of YY1 using the expression values from the NCI60 cell lines. Black and white bar represents the same distribution of probes, where a single black horizontal line indicates the presence of an YY1 binding motif (V$YY1_Q6) for that gene. (B) Among 2,537 genes that contain YY1 binding motifs, genes whose expression is correlated with that of YY1 are enriched for E2F binding sites (V$E2F1_Q3). (C) Heatmaps showing expression of putative downstream target genes of YY1 (bottom) and relation to taxane sensitivity in the NCI60 dataset (top). The top 150 YY1-positively correlated genes (Affymetrix U95Av2) were analyzed that contain both YY1 (V$YY1_Q6) and E2F (V$E2F1_Q3) binding motifs and that have at least one cell line with a log2(MAS5) expression value ≥7. Columns, individual cell lines; rows, individual gene probes. Cell lines (left to right): SNB-75, U251, A498, SF-295, UACC-62, SN12C, UO-31, 786-0, SNB-19, OVCAR-3, OVCAR-4, SK-MEL-28, UACC-257, RXF-393, SF-268, DU-145, SKOV-3, IGROV1, MALME-3M, OVCAR-8, SK-MEL-5, HOP-92, CAKI-1, TK-10, PC-3, T-47D, OVCAR-5, HCT-116, KM12, SW-620, HCT-15, MCF7, HT29, NCI-H226, MOLT-4, CCRF-CEM, NCI/ADR-RES, MDA-MB-231/ATCC, HCC-2998, A549/ATCC, NCIH522, NCI-H23, LOX IMVI, NCI-H460, EKVX, NCI-H322M, SK-MEL-2, SR, BT-549, HS 578T, SF-539, HOP-62, M14, MDA-MB-435, COLO 205, HL-60(TB), ACHN, RPMI-8226 and K-562. The ‘YY1 Low’ and ‘YY1 High’ clusters are indicated above the heat map. The four colored rows above the heat map represent GI50 values for (top to bottom) paclitaxel, docetaxel, cisplatin and carboplatin. Blue, resistant; red, sensitive.

Figure 3. Effect of YY1 knockdown on ovarian cancer cell behavior

(A, left) Efficiency of siRNA-mediated knockdown of YY1. Cells were transfected with control non-silencing siRNA oligos (C) or with two independent siRNA oligos specific to YY1 (1 and 2). Quantitative real-time RT-PCR assays were performed in duplicate and are shown for 72 hours post-transfection. Y-axis, expression (+SEM) relative to that of the control. (A, right) Western blotting of HEY and BG1 cells transfected with control or YY1- specific siRNAs at 48 and 72 hours post-transfection. GAPDH, endogenous control. (B) siRNA-mediated knockdown of YY1 inhibits cell proliferation. Y-axis, mean absorbance (+SEM) at 490 nm at 72 hours post-transfection. (C) Suppression of YY1/E2F target genes CDC6 and MCM5 measured by quantitative RT-PCR following knockdown of YY1. (D) Knockdown of YY1 inhibits anchorage-independent growth. Results are shown (+SEM) for 2×10³ seeded cells per well; proportional results were obtained when 5×10³ cells were seeded per well. (E) Knockdown of YY1 inhibits migration in wound healing assays. Photomicrographs were taken at the indicated time points following wounding.

Figure 4

Knockdown of YY1 leads to increased resistance to paclitaxel and docetaxel (A), but not to cisplatin (B) in ovarian cancer cell lines.