Analysis of TCGA data

RNA-seq and clinical data of breast cancer patients (n = 1075) were obtained from TCGA (https://portal.gdc.cancer.gov/), based on the access approval given to subscribed users, which does not require an ethics statement. Samples were stratified by expression of either ASAP1 (the gene encoding AMAP1) or PIAS3, with variable high/low thresholds. Samples with the top 15% ASAP1 expression was further stratified by relative expression levels of PIAS3, with the variable high/low thresholds. The average CA9 expression level (RSEM) was calculated in each group. Statistical significance was analyzed by the Brunner-Munzel test, which is independent of distributions and variances. For the analysis of the correlation of expression levels with prognosis, samples with top 15% ASAP1 expression and top 25% PIAS3 expression were categorized as the “ASAP1-high” and “PIAS3-high” groups, respectively. Survival curves were estimated based on the Kaplan-Meier method, and survival was compared by the log-rank test.