Gene signature associated with chemotherapy response in serous OvCa.

<p>(A) Identification of gene signature differentially expressed in chemoresistant and chemosensitive patients. (B) The selective genes (n = 227) distinguish the chemoresistant patients from the chemosensitive patients. (C) A predictive model on the basis of the gene signature reveals an accuracy of approximately 87.9% in correctly classifying chemoresistant and chemosensitive tumors (n = 232; green square = chemosensitive, blue triangle = chemoresistant). (D) An receiver operating characteristic (ROC) curve illustrates the predictive performance, with a sensitivity of approximately 95.2% and specificity of approximately 70% at the predictive score cutoff of approximately −0.16 that serves as a threshold for patient stratification in the TCGA data set. AUC: area under curve. (E) Pathway analysis shows that the gene signature is enriched in the morphologic function at cellular, tissue, and tumor levels. The dotted line denotes the cutoff for significance (<i>P</i> = 0.05). The shaded bars show the ratio of genes enriched in each function to the 227 genes.</p

Validation of gene signature.

<p>(A) The predictive model constructed from the TCGA training set was applied to an independent TCGA validation set (n = 261) and split the patients into two groups based on the score cutoff of −0.16 as determined by the ROC curve. Thirty five patients are identified to have an explicit response to chemotherapy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036383#pone.0036383-TCGA1" target="_blank">[22]</a>. (B) The two groups are well separated, with 212 patients in the low-scoring group and 49 in the high-scoring group. (C) Exclusion of patients with no survival data resulted in 109 patients in the low-scoring group and 29 in the high-scoring group. Kaplan-Meier analysis shows patients in the high-scoring group had poorer progression-free survival (<i>P</i> = 0.04). (D) The predictive model as applied to the external data set distinguishes the patients in the low-scoring group from in the high-scoring group; where the low-scoring group consists of the 70.1% patients (171 out of 244) with the highest predictive scores, and the high-scoring group consists of the 29.9% patients (73 out of 244) with the lowest predictive scores (see text for details). (E) Kaplan-Meier analysis shows patients in the high-scoring group had poorer progression-free survival than those in the low-scoring group (<i>P</i><0.0001).</p

Clinicopathologic characteristics of TCGA patients with serous OvCa that are used for tumor nuclear image profile and gene expression profile analyses.

<p>Abbreviations: FIGO, International Federation of Gynecology and Obstetrics; TCGA, The Cancer Genome Atlas; SD, standard deviation; WHO, World Health Organization.</p>¶<p>: Cases were staged according to the 1988 FIGO staging system.</p>ξ<p>: Surgical outcome was defined as the size of residual disease at the conclusion of the primary surgical procedure. This field was used to define surgical cytoreduction as optimal or suboptimal. Optimal was defined as no residual disease greater than 1 cm and included the variable categories of no macroscopic disease (<i>i.e.</i> microscopic residual disease) and 1 to 10 mm. Suboptimal was defined as residual disease greater than 1 cm and included the variable categories of 11 to 20 mm and greater than 20 mm.</p>ζ<p>: Local recurrence after the date of initial surgical resection.</p

Morphologically related genes at cellular, tissue, and tumor levels.

*<p>Fold difference in geometric means of chemoresistant tumors (numerator) compared with chemosensitive tumors (denominator).</p

Integrated analysis of morphologic features and gene signature.

<p>(A) Supervised analysis of gene expression data on the patients split by the Std_Ar_Bin2 feature values as described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036383#pone-0036383-g003" target="_blank">Figure 3B</a>. (B) Correlation of the highly correlated feature-gene pairs (<i>P</i><0.005), with negative correlations in green and positive correlations in red.</p

Bmi-1 knockdown perturbs the GSH biosynthesis pathway.

<p>The top panel represents total cellular GSH measured (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017918#s4" target="_blank">Materials and methods</a>) in ovarian cancer cells transfected with scrambled control or Bmi-1 siRNA treated with or without cisplatin for 24 h. The bottom panel represents fold change in gene expression (normalized with beta actin and compared to scrambled control) as determined by quantitative RT-PCR of ovarian cancer cells transfected with scrambled control or Bmi-1 siRNA for 48 h.</p

Effect of Bmi-1 knockdown on orthotopic chemoresistant ovarian cancer growth.

<p>To assess the effects of siRNA therapy on tumor growth, treatment was initiated 1 wk after i.p. injection (1.0×10⁶ CP20) of tumor cells. Mice were divided into four groups (n = 10 mice per group): (a) control siRNA-DOPC (150 µg/kg i.p. twice weekly), (b) control siRNA-DOPC + cisplatin (160 µg/mouse i.p. weekly), (c) Bmi-1 siRNA-DOPC (150 µg/kg i.p. twice weekly), and (d) Bmi-1 siRNA-DOPC + cisplatin (doses same as individual treatments). Treatment was continued until 4 weeks after tumor inoculation before sacrifice. (A) Total RNA was isolated from a portion of the tumor tissues and subjected to RT-PCR using primers for Bmi-1 and beta actin. The comparative C_t method was used to calculate the relative abundance of mRNA compared with that of beta actin expression. The experiment was performed in triplicate and significance determined using two-sided Student's t test, P<0.05 was considered significant. (B) Mouse and tumor weights and (C) the number of tumor nodules for each group were compared using Student's t test (for comparisons of two groups). A two-tailed P≤0.05 was deemed statistically significant.</p

Restoration of <i>let-7b</i> expression significantly reduces ovarian tumor growth <i>in vitro</i>.

<p>The <i>let-7b</i> mimic and control oligo (30 nM) were transfected into the A2780 (A), 2008 (B) and HOSE (C) cells by lipofectamine. The cell growth was monitored by a MTT assay.</p

Members of the <i>let-7</i> family show copy number deletions in medulloblastoma, breast, and ovarian cancers.

<p>Summary of DNA copy number alterations of the <i>let-7</i> family in 14 types of human cancers (n = 2,969). The <i>let-7</i> family is made up of thirteen members located at eight loci of the human genome. Many of them, such as <i>let-7a-1</i>, <i>let-7f-1</i> and <i>let-7d</i>, cluster together (A). Low q-values (upper threshold = 0.25) suggest that amplifications/deletions at this locus are significant and enriched by selective pressures. Dark green represents focal deletion of the <i>let-7</i> family.</p

Bmi-1 knockdown augments engagement of the DDR pathway in cisplatin treated ovarian cancer cells.

<p>(A) Ovarian cancer cells transfected with scrambled control or Bmi-1 siRNA were treated with or without cisplatin for 48 h. Western blot was performed for phospho Chk-2, total Chk-2, phospho-H2AX, total H2AX and beta actin using respective antibodies. (B) Scrambled control or Bmi-1 siRNA transfected CP-70 cells were subjected to confocal microscopy using 53BP1 antibody (red) and DAPI (blue nuclear staining) to demonstrate nuclear foci formation.</p