Yuehan Wu

MicroRNA-125a represses cell growth by targeting HuR in breast cancer

MicroRNAs (miRNAs) are a class of naturally occurring, small, non-coding RNAs that control gene expression during development, normal cell function, and disease. Although there is emerging evidence that some miRNAs can function as oncogenes or tumor suppressors, there is limited understanding of the role of miRNAs in cancer. In this study, we observed that the expression of miR-125a was inversely correlated with HuR expression in several different breast carcinoma cell lines. HuR is a stress-induced RNA binding protein whose expression is elevated or localization perturbed in several different cancers. Increased cytoplasmic localization of HuR is a prognostic marker in breast cancer. Real time PCR and gene reporter assays indicated that HuR was translationally repressed by miR-125a. Re-establishing miR-125a expression in breast cancer cells decreased HuR protein level and inhibited cell growth. Using MCF-7 breast cancer cells, we further clarified that miR-125a inhibited cell growth via a dramatic suppression of cell proliferation and promotion of apoptosis. In addition, cell migration was also inhibited by miR-125a overexpression. Importantly, the repression of cell proliferation and migration engendered by miR-125a was partly rescued by HuR re-expression. Our results suggest that miR-125a may function as a tumor suppressor for breast cancer, with HuR as a direct and functional target.

PARP1 is required for chromosomal translocations

Chromosomal translocations are common contributors to malignancy, yet little is known about the precise molecular mechanisms by which they are generated. Sequencing translocation junctions in acute leukemias revealed that the translocations were likely mediated by a DNA double-strand break repair pathway termed nonhomologous end-joining (NHEJ). There are major 2 types of NHEJ: (1) the classical pathway initiated by the Ku complex, and (2) the alternative pathway initiated by poly ADP-ribose polymerase 1 (PARP1). Recent reports suggest that classical NHEJ repair components repress translocations, whereas alternative NHEJ components were required for translocations. The rate-limiting step for initiation of alternative NHEJ is the displacement of the Ku complex by PARP1. Therefore, we asked whether PARP1 inhibition could prevent chromosomal translocations in 3 translocation reporter systems. We found that 2 PARP1 inhibitors or repression of PARP1 protein expression strongly repressed chromosomal translocations, implying that PARP1 is essential for this process. Finally, PARP1 inhibition also reduced both ionizing radiation-generated and VP16-generated translocations in 2 cell lines. These data define PARP1 as a critical mediator of chromosomal translocations and raise the possibility that oncogenic translocations occurring after high-dose chemotherapy or radiation could be prevented by treatment with a clinically available PARP1 inhibitor.

Mechanisms of oncogenic chromosomal translocations.

Chromosome translocations are caused by inappropriate religation of two DNA double‐strand breaks (DSBs) in heterologous chromosomes. These DSBs can be generated by endogenous or exogenous sources. Endogenous sources of DSBs leading to translocations include inappropriate recombination activating gene (RAG) or activation‐induced deaminase (AID) activity during immune receptor maturation. Endogenous DSBs can also occur at noncanonical DNA structures or at collapsed replication forks. Exogenous sources of DSBs leading to translocations include ionizing radiation (IR) and cancer chemotherapy. Spatial proximity of the heterologous chromosomes is also important for translocations. While three distinct pathways for DNA DSB repair exist, mounting evidence supports alternative nonhomologous end joining (aNHEJ) as the predominant pathway through which the majority of translocations occur. Initiated by poly (ADP‐ribose) polymerase 1 (PARP1), aNHEJ is utilized less frequently in DNA DSB repair than other forms of DSB repair. We recently found that PARP1 is essential for chromosomal translocations to occur and that small molecule PARP1 inhibitors, already in clinical use, can inhibit translocations generated by IR or topoisomerase II inhibition. These data confirm the central role of PARP1 in aNHEJ‐mediated chromosomal translocations and raise the possibility of using clinically available PARP1 inhibitors in patients who are at high risk for secondary oncogenic chromosomal translocations.

EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair.

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.

Microenvironmental control of the breast cancer cell cycle.

The mammary gland is one of the best‐studied examples of an organ whose structure and function are influenced by reciprocal signaling and communication between cells and their microenvironment. The mammary epithelial cell (MEC) microenvironment includes stromal cells and extracellular matrix (ECM). Abundant evidence shows that the ECM and growth factors co‐operate to regulate cell cycle progression, and that the ECM is altered in breast tumors. In particular, mammographically dense breast tissue is a significant risk factor for developing breast carcinomas. Dense breast tissue is associated with increased stromal collagen and epithelial cell content. In this article, we overview recent studies addressing the effects of ECM composition on the breast cancer cell cycle. Although the normal breast ECM keeps the MEC cycle in check, the ECM remodeling associated with breast cancer positively regulates the MEC cycle. ECM effects on the downstream biochemical and mechanosignaling pathways in both normal and tumorigenic MECs will be reviewed. Anat Rec, 2012.

Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5′ end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5′-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5′ end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.

The DNA repair component Metnase regulates Chk1 stability

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. Metnase (also termed SETMAR) is a SET histone methylase and transposase nuclease protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. We previously found that Chk1 phosphorylation of Metnase on S495 enhanced its DNA DSB repair activity but decreased its ability to re-start stalled replication forks. Here we show that phosphorylated Metnase feeds back to increase the half-life of Chk1. Chk1 half-life is regulated by DDB1 targeting it to Cul4A for ubiquitination and destruction. Metnase decreases Chk1 interaction with DDB1, and decreases Chk1 ubiquitination. These data define a novel pathway for Chk1 regulation, whereby a target of Chk1, Metnase, feeds back to amplify Chk1 stability, and therefore enhance replication fork arrest.

Cold-inducible RNA binding protein in mouse mammary gland development

RNA binding proteins (RBPs) regulate gene expression by controlling mRNA export, translation, and stability. When altered, some RBPs allow cancer cells to grow, survive, and metastasize. Cold-inducible RNA binding protein (CIRP) is overexpressed in a subset of breast cancers, induces proliferation in breast cancer cell lines, and inhibits apoptosis. Although studies have begun to examine the role of CIRP in breast and other cancers, its role in normal breast development has not been assessed. We generated a transgenic mouse model overexpressing human CIRP in the mammary epithelium to ask if it plays a role in mammary gland development. Effects of CIRP overexpression on mammary gland morphology, cell proliferation, and apoptosis were studied from puberty through pregnancy, lactation and weaning. There were no gross effects on mammary gland morphology as shown by whole mounts. Immunohistochemistry for the proliferation marker Ki67 showed decreased proliferation during the lactational switch (the transition from pregnancy to lactation) in mammary glands from CIRP transgenic mice. Two markers of apoptosis showed that the transgene did not affect apoptosis during mammary gland involution. These results suggest a potential in vivo function in suppressing proliferation during a specific developmental transition.

Abstract 2427: The 5′ endonuclease EEPD1 maintains genomic stability by mediating DNA repair pathway choice

EEPD1 (endonuclease/exonuclease/phosphatase family domain-containing 1) is an uncharacterized human protein that we found to be transcriptionally increased upon induction of DNA double strand breaks (DSBs). A549 cells with EEPD1 repressed by siRNA arrested at the G1/S and G2/M transitions and had markedly increased sensitivity to various DSB-inducing agents, such as camptothecin, VP-16, hydroxyurea (HU), and ionizing irradiation (IR). There are two major types of DSB repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), which is essential for replication fork repair. Using the EJ5 NHEJ reporter system, EEPD1 repression resulted in a 2-fold increase in NHEJ repair. However, formation of 53BP1 foci was unaffected in these cells, indicating that the initial step towards NHEJ was intact. However, in the HT256 HR reporter system, EEPD1 repression produced a 5-fold decrease in HR repair, indicating that EEPD1 mediates HR and inhibits NHEJ, placing it at the decision point for DSB repair. Immunofluorescence assays of HR components were performed in the presence of HU. EEPD1 repression significantly decreased γ-H2Ax, RAD51, and RPA32 foci formation, so placing EEPD1 above these HR components. The initial activity that commits a DSB to HR repair and away from NHEJ is 5′ end resection. This generates the 3′ ss DNA that results in ATR/Chk1 activation as well as RPA32 and γ-H2Ax foci. To assess this, a ss BRDU end resection assay was done after IR and there was a 4-fold decrease in 5′ end resection when EEPD1 was repressed. Consistently, there was also a decrease in ATR and Chk1 phosphorylation. Co-immunoprecipitation studies found that EEPD1 interacts with Ku80 and NBS1. In vitro assays using purified recombinant EEPD1 protein found its 5′ endonuclease activity was distinct from both Exo1 and DNA2, the two major mediators of 5′ end resection. This activity was, however, repressed by the Ku complex, indicating antagonism between EEPD1 and Ku. Cells with decreased EEPD1 expression showed severe nuclear anomalies, such as micronuclei, nucleoplasmic bridges that may have been caused by unopposed NHEJ when the functional HR was not available. Metaphase analysis in these cells also showed a significant increase in chromosomal aberrations, especially after IR and HU. These data suggest a novel model of collapsed replication fork repair pathway choice, where the free DS end binds the MRN complex, and NBS1 recruits EEPD1, inhibiting NHEJ and promoting HR via initiation of 5′ end resection. This is supported by the finding that EEPD1 is required for RPA32/ATRIP activation of ATR/Chk1, and all subsequent steps in repairing collapsed replication forks. Without such repair, the collapsed forks are end-joined to create chromosome fusions, which generate the nucleoplasmic bridging during mitosis. Lung and endometrial cancers have significant rates of EEPD1 mutation, further indicating that EEPD1 might be a novel tumor suppressor. Citation Format: Yuehan Wu, Suk-Hee Lee, Elizabeth A. Williamson, Brian L. Reinert, Gayathri Srinivasan, Sudha Singh, Aruna-Shanker Jaiswal, Silvia Tornaletti, Alexis C. Brantly, Robert A. Hromas. The 5′ endonuclease EEPD1 maintains genomic stability by mediating DNA repair pathway choice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2427. doi:10.1158/1538-7445.AM2014-2427