Supplementary Materials and Methods
Immunohistochemistry
TMA sections (4 µm) were de-waxed and placed through a graded alcohol series. Antigen retrieval was performed in high pH EnVision FLEX Target Retrieval Solution (Dako) in a pressure cooker for 2 mins. Endogenous peroxidase was blocked with EnVision FLEX Peroxidase-Blocking Reagent (Dako) before incubating sections with a rabbit polyclonal YAP (H-125) antibody (Santa Cruz Biotechnology) at 1:100 for 30 minutes at room temperature. Antigen-antibody complex was detected using Envision FLEX system (EnVision FLEX/HRP and EnVision FLEX DAB+ Chromogen (Dako). Slides were counterstained with hematoxylin, dehydrated, cleared and mounted.
Supplementary Results
Supplementary Figure 1. YAP stimulates anchorage-independent growth, migration and invasion of ovarian cancer cells.
(A) Expression levels of endogenous YAP (C), overexpressed wild-type YAP (Y), WW domain mutant YAP (W) and YAP-S127A (S) proteins in stably-infected OVCAR5 AND SKOV3 cells. (B and C) Quantitation of OVCAR5 (B) and SKOV3 (C) colonies expressing the indicated plasmids grown in soft agar. (D) Migration capacity in Transwell assays of OVCAR5 (D) and SKOV3 (E) cells expressing the indicated plasmids. (F and G) Invasive capacity in Matrigel invasion assays of OVCAR5 (F) and SKOV3 (G) cells expressing the indicated plasmids. Error bars in B-G represent standard deviation, n = 3, while statistically significant changes compared to control as determined by a t-test are denoted by * (P<0.05) or ** (p<0.01).
Supplementary Figure S2. Varying levels and subcellular localization of YAP protein in ovarian cancers.
Examples of cytoplasmic and nuclear YAP expression of different intensities that was observed in invasive serous cystadenocarcinomas of the ovary. Some tumors showed no YAP expression (o), whilst others showed weak staining (not shown). Cytoplasmic YAP staining varied from 1+ intensity (1) to 2+ intensity (2) and 3+ intensity (3). Nuclear YAP staining displayed a similar profile varying from 1+ intensity (1) to 2+ intensity (2) and 3+ intensity (3).
Supplementary Figure S3. Varying levels and subcellular localization of YAP protein in ovarian cancers, as well as percentage of cells stained.
The pie charts indicate the range of cytoplasmic and nuclear intensity of YAP protein detected. The bar graphs depict the fraction of cells stained positive for YAP in each tumor sample in either the cytoplasm or nucleus.
Supplementary Figure S4.
Gene expression heat map depicting expression levels of SWH pathway genes in five serous ovarian cancers with no YAP, and 19 serous ovarian cancers with high nuclear YAP. Red indicates higher expression, black indicates no change, whereas green indicates lowered expression. No significant changes in expression were observed in genes that have been validated as SWH pathway component in humans (A), or genes that potentially regulate the SWH pathway in mammals based on the fact that their Drosophila homologs have been characterized as regulators of the Drosophila SWH pathway (B).
Supplementary Figure S5. High levels of nuclear YAP protein expression correlates with poor prognosis of ovarian cancer.
Progression free survival of patients with ovarian cancer that were stratified in three groups based on immunohistochemical detection of YAP protein: low YAP (red), moderate YAP (green) and high nuclear YAP (black).
Supplementary Figure S6. Increased nuclear YAP protein expression correlates with poor prognosis of ovarian cancer.
Progression free survival of patients with ovarian cancer (a), serous invasive ovarian cancer (b), and endometrioid ovarian cancer (c), that were stratified in two groups based on immunohistochemical detection of YAP protein: low and moderate YAP, termed low YAP (red) and high nuclear YAP (green).
Supplementary Figure 7. YAP stimulates migration and anchorage-independent growth of ES-2 cells.
(A) Expression levels of endogenous YAP (C), overexpressed YAP-S127A (S), and Actin in ES-2 cells. (B) Migration capacity in Transwell assays of ES-2 cells expressing the indicated plasmids. (C) Soft agar assay with ES-2 cells expressing the indicated plasmids. Error bars in B and C represent standard deviation, n = 3. Statistically significant changes compared to control as determined by a t-test are denoted ** (p<0.01).
Supplementary Table S1
Raw YAP histopathological and AOCS clinical data used for all subsequent analyses.
Supplementary Table S2
Stratification of YAP histopathological data into Negative, Low, Moderate and High subgroups.
Supplementary Table S3
The top 1000 gene expression changes based on fold change that were observed between two classes of invasive serous ovarian tumors; a group of tumors that displayed no YAP expression (5 in total) and a group that displayed the highest levels of nuclear YAP (19 in total).
Supplementary Table S4
Candidate YAP target genes identified from microarray analysis of developing murine livers (1), murine NIH-3T3 cells (2) and human MCF10A cells (3, 4) that overexpress YAP.
Supplementary Table S5
Distribution of YAP expression (High or Low) in human ovarian cancer samples stratified based on histological subtype. Among the histological subtypes clear cell tumours displayed a higher proportion of samples with high nuclear YAP expression (Fisher’s exact test p-value = 0.013).
Supplementary Table S6
Association of YAP protein expression in different ovarian cancer histotypes with grade of disease (G1 = Grade1, G2 = Grade 2, G3 = Grade 3). No association was detected with grad in any subtype by Fisher’s exact test p-value = 0.013.
Supplementary Table S7
Number of samples, based on ovarian cancer molecular subtype (C1-C6), that showed low YAP or High YAP staining.
Supplementary References
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