Fig. S1. RNA-seq based RBP Library construction and CRISPR screen reveals RBP hits related to tumor growth and metastasis. Fig. S2. PHF5A is frequently up-regulated in breast cancer, associated with poor clinical outcome, and essential for cancer cell proliferation and migration. Fig. S3. PHF5A associates with the U2 snRNP complex and regulates complex stability. Fig. S4. PHF5A globally regulates splicing. Fig. S5. The PHD-like domain in PHF5A is required for its association with Histone H3. Fig. S6 PHF5A regulates FASTK splicing to antagonize apoptotic signaling in breast cancer cells. Table S1. Clinicopathologic Variables in 40 Cases of TNBC Patients. Table S2. Primers Used in RT-PCR and qPCR Analyses. Table S3. Gene List of a Census of 1,542 Manually Curated RBPs and 159 Library Contained RBPs. Table S4. sgRNA Sequence of Genes in RBP library. Table S5. RIGER Ranking of Screen Hits. Table S6. Correlation between Clinicopathologic Variables and Expression of PHF5A in 373 Cases of Breast Cancer. Table S7. Univariate and Multivariate Analysis of DFS. Table S8. Univariate and Multivariate Analysis of DFS in TNBC Subgroup.
ARTICLE ABSTRACT
Alternative splicing (AS) and its regulation play critical roles in cancer, yet the dysregulation of AS and its molecular bases in breast cancer development have not yet been elucidated. Using an in vivo CRISPR screen targeting RNA-binding proteins, we identified PHD finger protein 5A (PHF5A) as a key splicing factor involved in tumor progression. PHF5A expression was frequently upregulated in breast cancer and correlated with poor survival, and knockdown of PHF5A significantly suppressed cell proliferation, migration, and tumor formation. PHF5A was required for SF3b spliceosome stability and linked the complex to histones, and the PHF5A–SF3b complex modulated AS changes in apoptotic signaling. In addition, expression of a short truncated FAS-activated serine/threonine kinase (FASTK) protein was increased after PHF5A ablation and facilitated Fas-mediated apoptosis. This PHF5A-modulated FASTK–AS axis was widely present in breast cancer specimens, particularly those of the triple-negative subtype. Taken together, our findings reveal that PHF5A serves as an epigenetic suppressor of apoptosis and thus provides a mechanistic basis for breast cancer progression and may be a valuable therapeutic target.Significance: This study provides an epigenetic mechanistic basis for the aggressive biology of breast cancer and identifies a translatable therapeutic target. Cancer Res; 78(12); 3190–206. ©2018 AACR.
