Abstract P2-01-13: The splicing factor PHD finger protein 5A inhibits apoptosis to promote breast cancer progression

Abstract

Abstract Background: All the widely accepted hallmarks of cancer are known to be affected by aberrant splicing (AS), and splicing dysregulation itself is considered a valuable therapeutic target. Understanding the AS that promote cancer progression is crucial for the development of effective strategies for treating breast cancer. Methods: An in vivo CRISPR screen targeting RNA-binding proteins (RBPs) was performed to reveal the key splicing modulator (PHD finger protein 5A, PHF5A) of breast tumor progression. Immunohistochemistry method and survival analysis were performed using a tissue microarray (TMA) containing 450 breast carcinoma. Proliferation, transwell migration and in vivo tumor formation assays were utilized to assess the biological role of PHF5A. RNA sequencing and RT-PCR assay were used to identify PHF5A-regulated AS events in breast cancer cells. Biological functions and molecular pathways of the affected genes were investigated through a gene ontology (GO) analysis. Flow cytometry and Western blot analysis were used for apoptotic assessments. The correlation between PHF5A expression and AS events was further analysed using mRNA-Seq data of 40 paired breast cancer and adjacent normal breast tissues. And the correlation between the levels of PHF5A and cleaved caspase-3 were evaluated in the TMA. Results: According to RNA sequencing analysis of MCF10 cell series (MCF10A, MCF10AT, MCF10DCIS and MCF10CA1a), 159 RBPs were found to be up-regulated in cancer cells compared with non-cancer cells. And the CRISPR screen targeting these 159 RBPs systematically identified highest-ranking genes including PHF5A. In TMA cohort, high PHF5A expression was correlated with poor disease-free survival. PHF5A is frequently up-regulated in breast cancer and is essential for cancer cell proliferation, migration and tumor formation. Knockdown of PHF5A induces genome-wide AS events. The RT-PCR assay of MCF10CA1a cells showed that splicing changes of nine arbitrarily selected target genes were all modulated by PHF5A. GO analysis showed that PHF5A-regulated AS events were involved in apoptotic and anti-apoptotic pathways, among which FAS-activated serine/threonine kinase (FASTK) AS showed significant PSI (percent spliced in) difference. PHF5A knockdown appeared to switch full-length FASTK (FASTK-L) to an intron 5-retained variant (herein termed FASTK short, FASTK-S) in MCF10CA1a cells. The knockdown of PHF5A resulted in cleavage of caspase-3 and poly-ADP-ribose polymerase and conversion of the FASTK-L (61 kDa) and FASTK-S (42 kDa) proteins. Intriguingly, cells transduced with exogenous FASTK-S showed the most significant apoptotic effect, whereas the FASTK-L group presented a decreased apoptotic effect. The PHF5A ratios of paired non-tumor to tumor tissue were negatively correlated with the FASTK PSI differences between non-tumor and tumor tissues. A strong negative correlation was found between the PHF5A and cleaved caspase-3 levels in TMA. Conclusions: PHF5A depletion sensitizes cancer cells to apoptotic signaling partially through AS-mediated FASTK isoform conversion. This apoptotic suppressor plays a key role in breast cancer progression and acts as a prognostic indicator, and should be critically considered for optimization of the current therapeutic strategy. Citation Format: Zheng Y-Z, Xue M-Z, Shen H-J, Li X-G, Ma D, Gong Y, Hu X, Shao Z-M. The splicing factor PHD finger protein 5A inhibits apoptosis to promote breast cancer progression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-13.