Regulation of cyclin E1 by the breast cancer microenvironment

The behavior of breast epithelial cells is influenced by their microenvironment, which includes stromal cells and extracellular matrix. During breast cancer progression, the tissue microenvironment fails to control proliferation, resulting in uncontrolled growth and invasion. Upon invasion, the extracellular matrix encountered by breast cancer cells changes from primarily laminin and collagen IV to primarily collagen I. The current study used a three-dimensional (3D) collagen I culture model to address how the microenvironment controls the breast cancer cell cycle after invasion through the basement membrane. We show that culturing breast cancer cells in 3D collagen I inhibits proliferation through direct regulation of cyclin E1, a G1/S regulator that is overexpressed in breast cancer and implicated in its etiology. When the breast cancer cell line MDA-MB-231 was cultured within 3D collagen I gels, the G1/S transition was inhibited as compared to cells cultured on conventional 2D collagen or plastic dishes. Cells in 3D collagen downregulated cyclin E1 protein and mRNA, with no change in cyclin D1 level. Cyclin D1 regulates progression through early G1, where it is upregulated by signals from the extracellular environment. Cyclin D1 relocalized to the cytoplasm in 3D cultures and phosphorylation of Rb, a nuclear target for both cyclin E1- and cyclin D1-associated kinases, was decreased. Positive regulators of cyclin E1, the transcription factor c-Myc and cold-inducible RNA binding protein (CIRP), were decreased in 3D collagen cultures, while the collagen I receptor beta1 integrin was greatly increased. Inhibition of beta1 integrin function rescued proliferation, cyclin E1 and c-Myc expression, and Rb phosphorylation, but had no effect on cyclin D1 localization. We conclude that cyclin E1 is repressed independent of effects on cyclin D1 in a 3D collagen environment and dependent on beta1 integrin interaction with collagen I, reducing proliferation of invasive breast cancer cells. These results differ from studies of breast epithelial and cancer cells in 2D collagen or 3D Matrigel in which disruption of beta1 integrin function decreased proliferation. We also show that matrix metalloproteinases (MMPs) MMP-2 and MMP-9 increased dramatically in 3D collagen. Their inhibition reversed cyclin E1 reduction, indicating their upregulation may be key to this process. In addition to studying MDA-MB-231 cells, we show that 3D collagen has similar effects on the nontumorigenic breast epithelial cell line MCF10A and the breast cancer cell line HMT-3522 T4-2 cells. Proliferation and cyclin E1 were downregulated in both of these cell lines, with MCF10A cells undergoing global G1 arrest and subsequent apoptosis. Our data suggest that one way breast cancer cells adapt themselves to a collagen environment is by upregulating a collagen I receptor to decrease proliferation through cyclin E1 repression. Cyclin E1 therefore appears to link the ECM and cell cycle machinery, arguing the importance of considering the role of ECM in breast cancer etiology, valuing cyclin E1 as a prognostic factor, as well as targeting cyclin E1 overexpression in clinical therapy

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

The homologous recombination component EEPD1 is required for genome stability in response to developmental stress of vertebrate embryogenesis

Stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR), initiated by nuclease cleavage of branched structures at stalled forks. We previously reported that the 5' nuclease EEPD1 is recruited to stressed replication forks, where it plays critical early roles in HR initiation by promoting fork cleavage and end resection. HR repair of stressed replication forks prevents their repair by non-homologous end-joining (NHEJ), which would cause genome instability. Rapid cell division during vertebrate embryonic development generates enormous pressure to maintain replication speed and accuracy. To determine the role of EEPD1 in maintaining replication fork integrity and genome stability during rapid cell division in embryonic development, we assessed the role of EEPD1 during zebrafish embryogenesis. We show here that when EEPD1 is depleted, zebrafish embryos fail to develop normally and have a marked increase in death rate. Zebrafish embryos depleted of EEPD1 are far more sensitive to replication stress caused by nucleotide depletion. We hypothesized that the HR defect with EEPD1 depletion would shift repair of stressed replication forks to unopposed NHEJ, causing chromosome abnormalities. Consistent with this, EEPD1 depletion results in nuclear defects including anaphase bridges and micronuclei in stressed zebrafish embryos, similar to BRCA1 deficiency. These results demonstrate that the newly characterized HR protein EEPD1 maintains genome stability during embryonic replication stress. These data also imply that the rapid cell cycle transit seen during embryonic development produces replication stress that requires HR to resolve

Mendelian randomization supports genetic liability to hospitalization for COVID-19 as a risk factor of pre-eclampsia

BackgroundPre-eclampsia and eclampsia are among the major threats to pregnant women and fetuses, but they can be mitigated by prevention and early screening. Existing observational research presents conflicting evidence regarding the causal effects of coronavirus disease 2019 (COVID-19) on pre-eclampsia risk. Through Mendelian randomization (MR), this study aims to investigate the causal effect of three COVID-19 severity phenotypes on the risk of pre-eclampsia and eclampsia to provide more rigorous evidence.MethodsTwo-sample MR was utilized to examine causal effects. Summary-level data from genome-wide association studies (GWAS) of individuals of European ancestry were acquired from the GWAS catalog and FinnGen databases. Single-nucleotide polymorphisms associated with COVID-19 traits at p < 5 × −8 were obtained and pruned for linkage disequilibrium to generate instrumental variables for COVID-19. Inverse variance weighted estimates were used as the primary MR results, with weighted median and MR-Egger as auxiliary analyses. The robustness of the MR findings was also evaluated through sensitivity analyses. Bonferroni correction was applied to primary results, with a p < 0.0083 considered significant evidence and a p within 0.083–0.05 considered suggestive evidence.ResultsCritical ill COVID-19 [defined as hospitalization for COVID-19 with either a death outcome or respiratory support, OR (95% CI): 1.17 (1.03–1.33), p = 0.020] and hospitalized COVID-19 [defined as hospitalization for COVID-19, OR (95% CI): 1.10 (1.01–1.19), p = 0.026] demonstrated suggestive causal effects on pre-eclampsia, while general severe acute respiratory syndrome coronavirus 2 infection did not exhibit a significant causal effect on pre-eclampsia. None of the three COVID-19 severity phenotypes exhibited a significant causal effect on eclampsia.ConclusionsOur investigation demonstrates a suggestive causal effect of genetic susceptibility to critical ill COVID-19 and hospitalized COVID-19 on pre-eclampsia. The COVID-19 severity exhibited a suggestive positive dose–response relationship with the risk of pre-eclampsia. Augmented attention should be paid to pregnant women hospitalized for COVID-19, especially those needing respiratory support

Targeting oncogenic miR-335 inhibits growth and invasion of malignant astrocytoma cells

<p>Abstract</p> <p>Background</p> <p>Astrocytomas are the most common and aggressive brain tumors characterized by their highly invasive growth. Gain of chromosome 7 with a hot spot at 7q32 appears to be the most prominent aberration in astrocytoma. Previously reports have shown that microRNA-335 (miR-335) resided on chromosome 7q32 is deregulated in many cancers; however, the biological function of miR-335 in astrocytoma has yet to be elucidated.</p> <p>Results</p> <p>We report that miR-335 acts as a tumor promoter in conferring tumorigenic features such as growth and invasion on malignant astrocytoma. The miR-335 level is highly elevated in C6 astrocytoma cells and human malignant astrocytomas. Ectopic expression of miR-335 in C6 cells dramatically enhances cell viability, colony-forming ability and invasiveness. Conversely, delivery of antagonist specific for miR-335 (antagomir-335) to C6 cells results in growth arrest, cell apoptosis, invasion repression and marked regression of astrocytoma xenografts. Further investigation reveals that miR-335 targets disheveled-associated activator of morphogenesis 1(Daam1) at posttranscriptional level. Moreover, silencing of endogenous Daam1 (siDaam1) could mimic the oncogenic effects of miR-335 and reverse the growth arrest, proapoptotic and invasion repression effects induced by antagomir-335. Notably, the oncogenic effects of miR-335 and siDAAM1 together with anti-tumor effects of antagomir-335 are also confirmed in human astrocytoma U87-MG cells.</p> <p>Conclusion</p> <p>These findings suggest an oncogenic role of miR-335 and shed new lights on the therapy of malignant astrocytomas by targeting miR-335.</p

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

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

MoNuSAC2020:A Multi-Organ Nuclei Segmentation and Classification Challenge

Detecting various types of cells in and around the tumor matrix holds a special significance in characterizing the tumor micro-environment for cancer prognostication and research. Automating the tasks of detecting, segmenting, and classifying nuclei can free up the pathologists' time for higher value tasks and reduce errors due to fatigue and subjectivity. To encourage the computer vision research community to develop and test algorithms for these tasks, we prepared a large and diverse dataset of nucleus boundary annotations and class labels. The dataset has over 46,000 nuclei from 37 hospitals, 71 patients, four organs, and four nucleus types. We also organized a challenge around this dataset as a satellite event at the International Symposium on Biomedical Imaging (ISBI) in April 2020. The challenge saw a wide participation from across the world, and the top methods were able to match inter-human concordance for the challenge metric. In this paper, we summarize the dataset and the key findings of the challenge, including the commonalities and differences between the methods developed by various participants. We have released the MoNuSAC2020 dataset to the public