Abstract 1372: Novel role of the actin-binding factor Profilin-1 in DNA replication

Abstract

Abstract Introduction: Proper control of DNA replication is important for genome stability of all mitotic cells, and its deregulation leads to many forms of cancer. While the basic replication machinery and core regulatory factors have been thoroughly studied, new functional regulators continue to emerge. Here we present evidence for a novel function of the actin-binding protein profilin-1 (Pfn1) in DNA replication under both normal and stressed conditions. Pfn1 is essential for actin assembly, cell proliferation and survival, yet showing paradoxical antitumor activities for various types of cancer (breast, bladder, pancreatic, liver). It contains a unique binding site for poly-L-proline (PLP) motifs (XP≥5; X=A, L, I, G, S). Though predominantly cytoplasmic, it is also present in the cellular nucleus with poorly understood functions. Results: We find by chromatin fractionation that Pfn1 knockdown in the untransformed breast epithelial MCF-10A cells significantly inhibits the DNA-binding of many proteins in the replisome complex (e.g. PCNA, MCM-7, RFC, RPA, and DNA polymerase δ and ε) without affecting their cellular level. The knockdown specificity is confirmed by the phenotypic rescue via a Pfn1 construct containing RNAi-resistant silent mutations. In contrast, Pfn1 overexpression increases the DNA-binding of these proteins, and this requires its ability to bind PLPs since a PLP-binding defective Pfn1 mutant (S137D) shows no effect. Interestingly, Pfn1 undergoes robust nuclear translocation upon hydroxyurea (HU)-induced replication stress, and this occurs in various cell lines including MCF-10A and the breast cancer MCF-7 and MDA-MB-231 cells. Consistent with a role of Pfn1 in cellular response to stalled replication forks, its knockdown in HU-treated MCF-10A cells decreases ATR-dependent signaling (p-ATR, p-CHK1, p-P53, p-CDC2, p-RPA) but increases cellular survival, suggesting possible cell cycle checkpoint bypass and evasion of apoptotic cell death in the absence of Pfn1. Conclusions: By uncovering the effects of Pfn1 on DNA replication factors and cellular response to replication stress, we provide for the first time the compelling evidence for a “moonlighting” nuclear function of this well-known actin-binding protein that has been implicated in many forms of cancer. Our data suggest that nuclear Pfn1 is required for normal DNA replication, at least partially through its ability to secure the replisome complex at replication forks. Upon replication stress, more Pfn1 is translocated into the nucleus to facilitate ATR activation and proper DNA damage response (checkpoint activation, DNA repair, and apoptosis) which likely results from Pfn1's ability to stabilize the replication forks and the various associated proteins. Future work will be directed towards understanding the mechanistic details of Pfn1 involvement in DNA replication and translating this novel knowledge into anticancer treatments. Citation Format: Cuige Zhu, Sun-Joong Kim, Sean Holohan, Anna Rogers, Jieya Shao. Novel role of the actin-binding factor Profilin-1 in DNA replication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1372.